CN104370929B - A kind of method preparing Fugu ocellatus toxin - Google Patents
A kind of method preparing Fugu ocellatus toxin Download PDFInfo
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- CN104370929B CN104370929B CN201410573138.0A CN201410573138A CN104370929B CN 104370929 B CN104370929 B CN 104370929B CN 201410573138 A CN201410573138 A CN 201410573138A CN 104370929 B CN104370929 B CN 104370929B
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- fugu ocellatus
- ocellatus toxin
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- 241000054452 Takifugu ocellatus Species 0.000 title claims abstract description 62
- 239000003053 toxin Substances 0.000 title claims abstract description 62
- 231100000765 toxin Toxicity 0.000 title claims abstract description 62
- 238000000034 method Methods 0.000 title claims abstract description 26
- 239000007788 liquid Substances 0.000 claims abstract description 80
- 238000005342 ion exchange Methods 0.000 claims abstract description 28
- 238000002425 crystallisation Methods 0.000 claims abstract description 22
- 230000008025 crystallization Effects 0.000 claims abstract description 22
- 238000004062 sedimentation Methods 0.000 claims abstract description 18
- 238000004440 column chromatography Methods 0.000 claims abstract description 12
- 238000005189 flocculation Methods 0.000 claims abstract description 9
- 230000016615 flocculation Effects 0.000 claims abstract description 9
- 239000000243 solution Substances 0.000 claims description 53
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 52
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 48
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 45
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 42
- 239000006228 supernatant Substances 0.000 claims description 28
- 229910021529 ammonia Inorganic materials 0.000 claims description 24
- 239000000047 product Substances 0.000 claims description 24
- 239000002244 precipitate Substances 0.000 claims description 17
- 239000008367 deionised water Substances 0.000 claims description 16
- 229910021641 deionized water Inorganic materials 0.000 claims description 16
- 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 claims description 15
- 238000002791 soaking Methods 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 14
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 12
- 229910000831 Steel Inorganic materials 0.000 claims description 12
- 239000003480 eluent Substances 0.000 claims description 12
- 239000000178 monomer Substances 0.000 claims description 12
- 239000000741 silica gel Substances 0.000 claims description 12
- 229910002027 silica gel Inorganic materials 0.000 claims description 12
- 239000010959 steel Substances 0.000 claims description 12
- 239000008213 purified water Substances 0.000 claims description 10
- 239000003456 ion exchange resin Substances 0.000 claims description 9
- 229920003303 ion-exchange polymer Polymers 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- 238000004321 preservation Methods 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 238000005352 clarification Methods 0.000 claims description 8
- 239000000706 filtrate Substances 0.000 claims description 8
- 238000002347 injection Methods 0.000 claims description 8
- 239000007924 injection Substances 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- 238000004176 ammonification Methods 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 6
- 239000003729 cation exchange resin Substances 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 210000004185 liver Anatomy 0.000 claims description 6
- 238000010898 silica gel chromatography Methods 0.000 claims description 5
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 4
- 239000012141 concentrate Substances 0.000 claims description 4
- 238000007654 immersion Methods 0.000 claims description 4
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 claims description 4
- 229910000360 iron(III) sulfate Inorganic materials 0.000 claims description 4
- 239000012982 microporous membrane Substances 0.000 claims description 4
- 239000011347 resin Substances 0.000 claims description 4
- 229920005989 resin Polymers 0.000 claims description 4
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 3
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 3
- 229910052782 aluminium Inorganic materials 0.000 claims description 3
- 239000007791 liquid phase Substances 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 231100000331 toxic Toxicity 0.000 claims description 3
- 230000002588 toxic effect Effects 0.000 claims description 3
- 230000033228 biological regulation Effects 0.000 claims description 2
- 238000000703 high-speed centrifugation Methods 0.000 claims description 2
- 229920002401 polyacrylamide Polymers 0.000 claims description 2
- 238000001228 spectrum Methods 0.000 claims description 2
- 229960001866 silicon dioxide Drugs 0.000 description 10
- CFMYXEVWODSLAX-QOZOJKKESA-N tetrodotoxin Chemical compound O([C@@]([C@H]1O)(O)O[C@H]2[C@@]3(O)CO)[C@H]3[C@@H](O)[C@]11[C@H]2[C@@H](O)N=C(N)N1 CFMYXEVWODSLAX-QOZOJKKESA-N 0.000 description 8
- 229950010357 tetrodotoxin Drugs 0.000 description 8
- CFMYXEVWODSLAX-UHFFFAOYSA-N tetrodotoxin Natural products C12C(O)NC(=N)NC2(C2O)C(O)C3C(CO)(O)C1OC2(O)O3 CFMYXEVWODSLAX-UHFFFAOYSA-N 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 6
- 238000011953 bioanalysis Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 239000002253 acid Substances 0.000 description 3
- 229960000935 dehydrated alcohol Drugs 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 235000003891 ferrous sulphate Nutrition 0.000 description 3
- 239000011790 ferrous sulphate Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 238000004080 punching Methods 0.000 description 3
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N anhydrous guanidine Natural products NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 150000003839 salts Chemical group 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- LFXVBFTVUBWIQT-UHFFFAOYSA-N 3,4,4a,5,6,7,8,8a-octahydro-2h-quinazolin-1-amine Chemical class C1CCCC2N(N)CNCC21 LFXVBFTVUBWIQT-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010012335 Dependence Diseases 0.000 description 1
- 208000018526 Narcotic-Related disease Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 239000004411 aluminium Substances 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000001949 anaesthesia Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 125000002837 carbocyclic group Chemical group 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000003311 flocculating effect Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 230000009965 odorless effect Effects 0.000 description 1
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 description 1
- 201000005040 opiate dependence Diseases 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 150000002905 orthoesters Chemical group 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical class OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- 230000005588 protonation Effects 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000009967 tasteless effect Effects 0.000 description 1
- 230000005760 tumorsuppression Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The invention provides a kind of method preparing Fugu ocellatus toxin, specifically include following steps: soak；Sedimentation；Flocculation；Ion exchanges；Low pressure column chromatography；Performance liquid chromatographic column；Crystallization.The present invention use above steps before and after closely coupled, interlock layer by layer, preferably Fugu ocellatus toxin and 4 table Fugu ocellatus toxin can be separated, and make the yield of Fugu ocellatus toxin be greatly improved, the high purity more than 99% of the Fugu ocellatus toxin prepared.
Description
Technical field
The invention belongs to field of medicine preparations, particularly to a kind of method preparing Fugu ocellatus toxin.
Background technology
Fugu ocellatus toxin (tetrodotoxin) is a kind of amino perhydro quinazoline type compound, tool
There is cage modle ortho esters structure, for white crystals, odorless, tasteless, be slightly soluble in water, insoluble in organic
Solvent；The architectural feature of Fugu ocellatus toxin is to have 1 carbocyclic ring, 1 guanidine radicals, 6 hydroxyls,
There is the separate ring that one and half aldoniolactones are connected to C-5 and C-10 position.It is the most special
A kind of organic compound, intramolecular guanidine radicals and nitrogen-atoms are protonations, positive carbonyl acid also from
Solving is anion, so Fugu ocellatus toxin is to exist with inner salt form.Fugu ocellatus toxin
(tetrodotoxin) there is treating narcotic addiction and the clinical effectiveness of the not addiction of easing pain in clinic, also have it
Blood pressure lowering, local anaesthesia, effect of aspect of tumor suppression.
The Fugu ocellatus toxin (tetrodotoxin) content in protozoa is little, typically million
Divide on this order of magnitude of 0.1-5, so the amount of pre-treatment is very big in extraction, and Fugu ocellatus
Toxin has substantial amounts of free amino acid, peptides, fat, albumen, colloidal substance, nothing after soaking
The non-targeted products such as machine salt are present in soak, domestic and international researcher the most all use by
Soak heated and boiled impurity degeneration precipitates, then passes through quickly cooling, the side settling, filtering or be centrifuged
Method removes the precipitated impurities such as the albumen in soak.But owing to wanting heated and boiled in the method,
And Fugu ocellatus toxin is to thermoae instability, heat loss 11.8% in 30 minutes when 80 DEG C, at 90 DEG C
Shi Jiare loss 31.2% in 30 minutes, the most not only causes the loss of Fugu ocellatus toxin, and
And reduce the yield of Fugu ocellatus toxin.
Extraction separation to Fugu ocellatus toxin (tetrodotoxin) has a lot of relevant report both at home and abroad,
The Fugu ocellatus toxin of researcher extraction both at home and abroad is substantially by alumina column chromatography, filter paper layer
Analysis, ion exchange and carbon post absorption, the most useful two step resins exchange obtain Fugu ocellatus toxin
(tetrodotoxin) coarse crystallization, then with picric acid salt forming method recrystallization repeatedly, thus obtain
Pure Fugu ocellatus toxin crystallization, purity is about 95%, but the method can not completely by with Fugu ocellatus toxin
Its isomer 4-table Fugu ocellatus toxin (tetrodotoxin) that character is the most close is separated.
Summary of the invention
The technical problem to be solved is to provide a kind of yield improving Fugu ocellatus toxin
And can preferably Fugu ocellatus toxin and 4-table Fugu ocellatus toxin be separated prepare Fugu ocellatus toxin
Method.
The invention provides a kind of method preparing Fugu ocellatus toxin, specifically include following steps:
Step one: soak
Ocellated puffer liver or roe are cut into inch strips or after block, soaks by deionized water or purified water,
To feed liquid, be dipped to feed liquid the most toxic till；
Step 2: sedimentation
The feed liquid of step one gained is placed in 6～10 DEG C of sedimentations place 10～12 hours, separates
Supernatant, and it is mixed to get clear liquor with supernatant again after centrifugal for precipitate also filter cleaner；
Step 3: flocculation
The clear liquor of step 2 gained ammonia spirit is adjusted pH to 7～9, adds flocculant molten
Liquid regulation pH to 4～6, stirs evenly, standing sedimentation, then separates supernatant, and by precipitate
Mix with supernatant again after high speed centrifugation filter cleaner；
Step 4: ion exchanges
Step 3 is mixed the feed liquid of gained, upper ion exchange column, exchanges to pH6.5～7.0,
Exchange rushes post extremely clarification by deionized water or purified water after terminating, then with acetum eluting,
Collect the component containing target product, adjust pH to 5～7 with ammonia spirit, be concentrated under reduced pressure to give dense
Contracting liquid.
Step 5: low pressure column chromatography
Concentrated solution and the dry silica gel of step 4 gained being mixed, upper Lower pressure silica gel chromatography column separates,
And add and consist of the eluent of n-butyl alcohol, acetic acid and water and carry out eluting, collect containing target product
Effluent part, be filtrated to get filtrate, be concentrated under reduced pressure to give concentrated solution.
Step 6: performance liquid chromatographic column
By the performance liquid chromatographic column after step 5 gained concentrated solution injection balance, with acetum
Eluting, collects the effluent concentrating under reduced pressure containing target product, obtains concentrated solution.
Step 7: crystallization
The concentrated solution of step 6 gained ammonia spirit is adjusted pH to 8～9, cold preservation, separates out knot
Crystalline substance, leaches, and with absolute ethanol washing, room temperature drying under reduced pressure i.e. obtains the crystallization of Fugu ocellatus toxin monomer.
In described step one, described ocellated puffer liver or roe be cut into 1～2cm bar or block,
Put and rustless steel soaking compartment add 1～the deionized water of 2 times or purified water are 6～10 DEG C of immersions,
And stir, after 18～24 hours, feed liquid is discarded to storage from the end opening of rustless steel soaking compartment. in good time
In hopper or settling tank.
In described step 3, it is that the clear liquor of step 2 gained is adjusted pH with the ammonia spirit of 25%
To 7～9, add flocculant solution to pH4～6, the speed with 50～300rpm/min stir 5～
10min, standing sedimentation 18～24 hours at temperature is 6～10 DEG C, after separating supernatant,
By precipitate through high speed centrifuge, centrifugal under rotating speed is 5000～10000rpm/min
20～40 minutes, and mix with supernatant again after filter cleaner.
Described flocculant is bodied ferric sulfate, polyaluminium sulfate, polyacrylamide or polyaluminium
Aluminum, injected volume is 0.01～5g/L.
In described step 4, be the feed liquid that step 3 is mixed gained, on rush through ammonification and with water
To the ion exchange column of pH8～9, exchange is to pH6.5～7.0, and wherein feed liquid and ion exchange
Amount ratio between resin is 40～60mL/g, and exchange is rushed by deionized water or purified water after terminating
Post is to clarification, then with 5～the acetum eluting of 10%, and acetum and upper feed liquid
Volume ratio is 1:90～180, collects the component containing target product, adjusts pH to 5～7 with ammonia,
At 0.9～1.0 atmospheric pressure concentrating under reduced pressure, concentrate 90～100 times, obtain concentrated solution.
The ion exchange resin used by ion exchange column in described step 4 is D152 weak-type
Macroporous acrylic cation exchange resin.
In described step 5, it is by the concentrated solution of step 4 gained, and dry silica gel is mixed, both
The ratio of amount be 0.8～1mL/g, upper Lower pressure silica gel chromatography column separates, and pressure is 0.7～0.9
Individual atmospheric pressure, addition consists of the eluent of n-butyl alcohol, acetic acid and water and carries out eluting, eluent
Being 50:1 with the volume ratio of upper feed liquid, wherein the volume ratio of n-butyl alcohol, acetic acid and water is respectively
For 5:3:1, collect the effluent part containing target product, with 0.45 μm filtering with microporous membrane,
Obtain filtrate with 0.9～1.0 atmospheric pressure, 90～100 times of concentrating under reduced pressure, obtain concentrated solution.
In described step 6, be by step 5 gained concentrated solution injection balance after high-efficient liquid phase color
Spectrum post, performance liquid chromatographic column is C18Post, applied sample amount is 0.2～0.4mL, with volume basis
Than being the acetum eluting of 0.4～0.6%, the volume ratio of eluent and upper feed liquid be 80～
150:1, monitors with differential refraction monitor, collect the effluent containing target product with 0.9～
1.0 atmospheric pressure, 90～100 times of concentrating under reduced pressure, obtain concentrated solution.
In described step 7, it is to be 8～10% by the concentrated solution volume fraction of step 6 gained
Ammonia adjust pH to 8～9, cold preservation, separate out crystallization, leach, with absolute ethanol washing,
-1.1～-0.9 atmospheric pressure under, normal temperature drying 24～within 48 hours, i.e. obtain Fugu ocellatus toxin monomer knot
Brilliant.
The present invention use immersion, settle, flocculate, ion exchange, low pressure column chromatography, high-efficient liquid
The step of phase chromatograph and crystallization is used for preparing Fugu ocellatus toxin, closely coupled before and after each step, layer by layer
Interlock, preferably Fugu ocellatus toxin can be separated with 4-table Fugu ocellatus toxin, and make tetrodotoxin, TTX
The yield of element is greatly improved, the high purity more than 99% of the Fugu ocellatus toxin prepared.
Detailed description of the invention
The invention provides a kind of method preparing Fugu ocellatus toxin, be by ocellated puffer liver or roe solution
The low temperature that adds water after freezing soaks, and settles overnight, after separating supernatant and adding flocculant flocculation, at a high speed
Centrifugal carry out ion exchange again, low pressure column chromatography, through performance liquid chromatographic column, concentrating under reduced pressure,
Alkali tune, cold preservation, leach, wash and drying under reduced pressure i.e. obtains Fugu ocellatus toxin (tetrodotoxin)
Monomer crystallizes.
Specifically include following steps:
Step one: soak
It is cut into bar or the block of 1～2cm after ocellated puffer liver or roe being thawed, puts rustless steel and soak
Groove adds 1～the deionized water of 2 times or purified water is 6～10 DEG C of immersions, and stir in good time,
After 18～24 hours, feed liquid is discarded to stock chest or settling tank from the end opening of rustless steel soaking compartment
In, dip operation repeat 3～4 times, be dipped to feed liquid the most toxic till.
Step 2: sedimentation
The feed liquid of step one gained is placed in 6～10 DEG C of sedimentations place 10～12 hours, separates
Supernatant, and it is mixed to get clear liquor with supernatant again after centrifugal for precipitate also filter cleaner.
Step 3: flocculation
The clear liquor of step 2 gained 25% (v/v) ammonia spirit is adjusted pH to 7～9, adds
5～the flocculant solution appropriate (injected volume is 0.01～5g/L) of 30% (m/v) to pH4～6,
Speed with 50～300rpm/min stirs 5～10min, stands at temperature is 6～10 DEG C
Settle 18～24 hours.
By flocculation by albumen substantial amounts of in solution, polypeptide, colloid, etc. macromolecular substances assemble
Settle down, in case removing.Described flocculant be bodied ferric sulfate, polyaluminium sulfate, poly-third
Acrylamide or aluminium polychlorid, more preferably bodied ferric sulfate.
Feed liquid after flocculating sedimentation is separated supernatant, and by precipitate through high speed centrifuge,
Under rotating speed is 5000～10000rpm/min behind centrifugal 20-40 minute, and filter cleaner again
Mix with supernatant.
Step 4: ion exchanges
Step 3 is mixed the feed liquid of gained, on through ammonification and with water punching to the ion of pH8～9
Exchange column, exchange is to pH6.5～7.0, and wherein the amount ratio between feed liquid and ion exchange resin is
40～60mL/g, coordinate with bioanalysis (mice) or instrumental method and monitor whether ion exchange completely,
Exchange rushes post to clarification, then with 5～10% (v/v) by deionized water or purified water after terminating
Acetum eluting, collect containing the component of target product, adjust pH to 5～7 with ammonia spirit,
At 0.9～1.0 atmospheric pressure concentrating under reduced pressure, concentrate 90～100 times, obtain concentrated solution.
Ion exchange resin used by described ion exchange column is preferably D152 weak-type macropore third
Olefin(e) acid cation exchange resin (NH4 +)。
Step 5: low pressure column chromatography
By the concentrated solution of step 4 gained, and dry silica gel is mixed, the ratio of both amounts be 0.8～
1mL/g, upper Lower pressure silica gel chromatography column separates, and pressure is 0.7～0.9 atmospheric pressure, addition group
Become the eluent of n-butyl alcohol, acetic acid and water and carry out eluting, wherein n-butyl alcohol, acetic acid and water
Volume ratio is respectively 5:3:1, coordinates monitoring with bioanalysis (mice), collects and produces containing target
The effluent part of thing, with 0.45 μm filtering with microporous membrane, obtains filtrate with 0.9～1.0
Atmospheric pressure, 90～100 times of concentrating under reduced pressure, obtain concentrated solution.
Use dry silica gel be filler carry out low pressure column chromatography method can be miscellaneous by aminoacid, inorganic salt etc.
Mass-energy is the most preferably separated off with Fugu ocellatus toxin, carries out low pressure column chromatography than selecting other filler
Method good separating effect, used time short and easy grasp.
Step 6: performance liquid chromatographic column
By the performance liquid chromatographic column (ODS) after step 5 gained concentrated solution injection balance, high
It is 5 μm, internal diameter 20mm and length 250mm that effect liquid phase chromatogram post (ODS) is preferably granularity
C18Post, each applied sample amount is 0.2～0.4mL, containing about Fugu ocellatus toxin 30ug-60ug, with body
Long-pending percentage ratio is the acetum eluting of 0.4～0.6%, monitors with differential refraction monitor, receives
Collection containing the effluent of target product with 0.9～1.0 atmospheric pressure, 90～100 times of concentrating under reduced pressure,
Obtain concentrated solution.
Step 7: crystallization
By the concentrated solution volume fraction of step 6 gained be 8～10% ammonia spirit adjust pH extremely
8～9, cold preservation, separate out crystallization, leach, with absolute ethanol washing ,-1.1～-0.9
Under atmospheric pressure, normal temperature drying 24～within 48 hours, i.e. obtain Fugu ocellatus toxin monomer crystallization.
With specific embodiment, the present invention is described in detail below.
Embodiment 1
Present embodiments provide a kind of method preparing Fugu ocellatus toxin, specifically include following steps:
Step one: soak
Take bar or the block being cut into 1～2cm after roe 25kg thaws, be placed in rustless steel soaking compartment,
Adding deionized water at 8 DEG C to soak 4 times, solid-liquid ratio is followed successively by 2:1,1:1,1:1,1:0.5,
Every stirring in 2 hours once, material is released to obtain from the end opening of rustless steel soaking compartment after soaking 24 hours
Liquid 110kg pumps in settling tank.
Step 2: sedimentation
The feed liquid of step one gained is placed in 8 DEG C settle 11 hours, separates supernatant, and will
Precipitate is centrifugal and filters, again with upper after slagging-off with the fast grade filter paper that aperture is 80～100 microns
Clear liquid is mixed to get clear liquor 108kg.
Step 3: flocculation
The clear liquor of the step 2 gained ammonia spirit of 25% (v/v) is adjusted pH to 8.3,
Mixing, add 20% (m/v) polymeric ferrous sulphate solution 1500mL regulate pH to 5, with
The speed stirring 7min of 200rpm/min, standing sedimentation 20 hours at temperature is 8 DEG C, material
Liquid layering separates out a large amount of precipitate, separates supernatant 65kg, and by precipitate through at a high speed from
Scheming, is 5000～10000rpm/min to be centrifuged 30 minutes with rotating speed, with aperture be 80～
Mix with supernatant again after the fast grade filter paper filter cleaner of 100 microns, there are feed liquid 105kg.
Step 4: ion exchanges
By the feed liquid of step 3 gained, above shift to an earlier date through ammonification and hand over the ion of water punching to pH8.5
Changing post, the ion exchange resin used by described ion exchange column is D152 weak-type macropore propylene
Weak acid cation exchange resin (NH4 +), the amount between feed liquid and ion exchange resin than 50mL/g,
After exchange pH to 6.5, coordinate with bioanalysis (mice) and monitor whether ion exchange completely,
Exchange rushes post extremely clarification with deionized water after terminating, molten with the acetic acid that percentage by volume is 7.5%
Liquid carries out eluting, collects the component containing target product, obtains feed liquid 1500mL, with 9% (v/v)
Ammonia spirit adjust pH to 5.5, under 0.95 atmospheric pressure concentrate 95 times, obtain concentrated solution.
Step 5: low pressure column chromatography
By the concentrated solution of step 4 gained, mix by 0.9mL/g and dry silica gel, upper low pressure silica gel
Post separates, and pressure is 0.9 atmospheric pressure, adds and consists of n-butyl alcohol, acetic acid and water
Eluent carries out eluting, and wherein the volume ratio of n-butyl alcohol, acetic acid and water is respectively 5:3:1,
Coordinate monitoring with bioanalysis (mice), collect the effluent part containing target product, obtain feed liquid
980mL, with 0.45 μm filtering with microporous membrane, obtain filtrate with 0.95 atmospheric pressure, 95 times
Concentrating under reduced pressure, obtains concentrated solution.
Step 6: performance liquid chromatographic column
By the performance liquid chromatographic column (ODS) after step 5 gained concentrated solution injection balance, described
Performance liquid chromatographic column is C18Post, each applied sample amount is 0.3mL (containing about Fugu ocellatus toxin 45ug),
With the acetum eluting that percent by volume is 0.5%, monitor with differential refraction monitor, receive
The collection effluent 80mL containing target product, at 0.95 atmospheric pressure, 95 times of concentrating under reduced pressure, obtains
Concentrated solution.
Step 7: crystallization
The ammonia spirit that concentrated solution percent by volume is 9% of step 6 gained is adjusted pH to 9,
Place 24 hours in 4OC cold preservation, separate out crystallization, with the middling speed filter that aperture is 30～50 microns
Paper leaches, and divides 3 washings, under-1.0 atmospheric pressure, room temperature with 10 times amount dehydrated alcohol
It is dried 48 hours, obtains Fugu ocellatus toxin monomer crystallization 320mg.
The purity crystallized through high performance liquid chromatography detection gained Fugu ocellatus toxin monomer is 99.40%.
Embodiment 2
Present embodiments provide a kind of method preparing Fugu ocellatus toxin, specifically include following steps:
Step one: soak
Take roe 200kg, be cut into bar or the block of 1～2cm after defrosting, put rustless steel soaking compartment
In, adding deionized water at 6 DEG C and soak 4 times, solid-liquid ratio is followed successively by 2:1,1:1,1:1,1:0.5,
Every stirring in 2 hours once, material is released to obtain from the end opening of rustless steel soaking compartment after soaking 24 hours
Liquid 900kg pumps in settling tank.
Step 2: sedimentation
The feed liquid of step one gained is placed in 6 DEG C settle overnight 10 hours, separates supernatant,
And by centrifugal for precipitate and with after the fast grade filter paper filter cleaner that aperture is 80～100 microns again
It is mixed to get clear liquor 860kg with supernatant.
Step 3: flocculation
The clear liquor of the step 2 gained ammonia spirit of 25% (v/v) is adjusted pH to 7, mixed
Even, add 20% (m/v) polymeric ferrous sulphate solution 43000mL regulate pH to 5, with
The speed stirring 6min of 300rpm/min, standing sedimentation 24 hours at temperature is 8 DEG C, material
Liquid layering separates out a large amount of precipitate, separates supernatant 520kg, and by precipitate in batches through too high
Speed centrifuge, is 5000～10000rpm/min to be centrifuged 20 minutes with rotating speed, with aperture is
Mix with supernatant again after the fast grade filter paper filter cleaner of 80～100 microns, there are feed liquid
840kg。
Step 4: ion exchanges
By the feed liquid of step 3 gained, above shift to an earlier date through ammonification and exchange with the ion of water punching to pH8
Post, the ion exchange resin used by described ion exchange column is D152 weak-type macroporous acrylic
Cation exchange resin (NH4 +), the amount between feed liquid and ion exchange resin, than 40mL/g, is handed over
Shift to pH6.5, coordinate with bioanalysis (mice) and monitor whether ion exchange completely, exchange knot
Shu Houyong deionized water rushes post extremely clarification, washes with the acetum that percentage by volume is 6%
De-, collect the component containing target product, obtain feed liquid 2.52kg, be the ammonia of 9% with volume fraction
Aqueous solution adjusts pH to 6, and under 0.9 atmospheric pressure, 100 times of concentrating under reduced pressure, obtain concentrated solution.
Step 5: low pressure column chromatography
By the concentrated solution of step 4 gained, ratio and dry silica gel in 0.8mL/g are mixed, upper low
Pressure silicagel column separates, and pressure is 0.7 atmospheric pressure, adds and consists of n-butyl alcohol, acetic acid
Carrying out eluting with the eluent of water, wherein the volume ratio of n-butyl alcohol, acetic acid and water is respectively 5:
3:1, collects the effluent part containing target product, obtains feed liquid 10.8kg, micro-by 0.45 μm
Hole membrane filtration, obtains filtrate with 90 times of concentrating under reduced pressure of 0.9 atmospheric pressure, obtains concentrated solution.
Step 6: through performance liquid chromatographic column
By the performance liquid chromatographic column (ODS) after step 5 gained concentrated solution injection balance, described
Performance liquid chromatographic column is C18The each applied sample amount of post is 0.2mL (containing about Fugu ocellatus toxin 30ug),
With the acetum eluting that percent by volume is 0.4%, monitor with differential refraction monitor, receive
The collection effluent 800mL containing target product, 100 times of concentrating under reduced pressure of 0.9 atmospheric pressure, obtain
Concentrated solution.
Step 7: crystallization
The ammonia spirit that concentrated solution volume fraction is 9% of step 6 gained is adjusted pH to 9,
Place 24 hours in 4OC cold preservation, separate out crystallization, with the middling speed filter that aperture is 30～50 microns
Paper leaches, and divides 3 washings, under-1.0 atmospheric pressure, room temperature with 10 times amount dehydrated alcohol
It is dried 48 hours, obtains Fugu ocellatus toxin monomer crystallization 2.68g.
The purity crystallized through high performance liquid chromatography detection gained Fugu ocellatus toxin monomer is 99.60%.
Embodiment 3
Present embodiments provide a kind of method preparing Fugu ocellatus toxin, specifically include following steps:
Step one: soak
Take roe 200kg, be cut into bar or the block of 1～2cm after defrosting, put rustless steel soaking compartment
In, adding deionized water at 10 DEG C and soak 4 times, solid-liquid ratio is followed successively by 2:1,1:1,1:1,1:0.5,
Every stirring in 4 hours once, material is released to obtain from the end opening of rustless steel soaking compartment after soaking 24 hours
Liquid 900kg pumps in settling tank.
Step 2: sedimentation
The feed liquid of step one gained is placed in 10 DEG C settle overnight 12 hours, separates supernatant,
And by centrifugal for precipitate and with after the fast grade filter paper filter cleaner that aperture is 80～100 microns again
It is mixed to get clear liquor 880kg with supernatant.
Step 3: flocculation
The clear liquor of step 2 gained 25% (v/v) ammonia spirit is adjusted to pH9, mixing,
The polymeric ferrous sulphate solution 31000mL adding 30% regulates to pH5, with the speed of 300rpm/min
Stirring 6min, standing sedimentation 24 hours at temperature is 10 DEG C, feed liquid layering separates out a large amount of heavy
Shallow lake thing, separates supernatant 520kg, and by precipitate in batches through high speed centrifuge, with rotating speed
It is 5000～10000rpm/min to be centrifuged 40 minutes, is 80～100 microns quick with aperture
Mix with supernatant again after filter paper filtering slagging-off, there are feed liquid 850kg.
Step 4: ion exchanges
By the feed liquid of step 3 gained, upper ion exchange column, used by described ion exchange column from
Sub-exchange resin is D152 weak-type macroporous acrylic cation exchange resin (NH4 +), feed liquid and
Amount between ion exchange resin is than 60mL/g, and ion exchange column shifts to an earlier date through ammonification and rushes extremely with water
PH9, exchanges to pH7, coordinates with bioanalysis (mice) and monitor whether ion exchange completely,
Exchange rushes post extremely clarification with deionized water after terminating, and is the acetum of 10% with percentage by volume
Carry out eluting, collect the component containing target product, obtain feed liquid 1.52kg, with percent by volume
It is the ammonia spirit tune pH to 6 of 9%, at 90 times of concentrating under reduced pressure of 0.95 atmospheric pressure, obtains dense
Contracting liquid.
Step 5: low pressure column chromatography
By the concentrated solution of step 4 gained, ratio and dry silica gel in 1.0mL/g are mixed, upper low
Pressure silicagel column separates, and pressure is 0.9 atmospheric pressure, adds and consists of n-butyl alcohol, acetic acid
Carrying out eluting with the eluent of water, wherein the volume ratio of n-butyl alcohol, acetic acid and water is respectively 5:
3:1, collects the effluent part containing target product, obtains feed liquid 10.2kg, micro-by 0.45 μm
Hole membrane filtration, obtains filtrate with 90 times of concentrating under reduced pressure of 1.0 atmospheric pressure, obtains concentrated solution.
Step 6: through performance liquid chromatographic column
By the performance liquid chromatographic column (ODS) after step 5 gained concentrated solution injection balance, described
Performance liquid chromatographic column is C18The each applied sample amount of post is 0.4mL (containing about Fugu ocellatus toxin 60ug),
With the acetum eluting that percent by volume is 0.6%, monitor with differential refraction monitor, receive
The collection effluent 600mL containing target product, with 90 times of concentrating under reduced pressure of 1.0 atmospheric pressure,
To concentrated solution.
Step 7: crystallization
The ammonia spirit that concentrated solution volume fraction is 9% of step 6 gained is adjusted to pH9,
4 DEG C of cold preservations are placed 24 hours, separate out crystallization, with the Medium speed filter paper that aperture is 30～50 microns
Leach, divide 3 washings, under-1.0 atmospheric pressure, air setting with 10 times amount dehydrated alcohol
Dry 48 hours, obtain Fugu ocellatus toxin monomer crystallization 2.75g.
The purity crystallized through high performance liquid chromatography detection gained Fugu ocellatus toxin monomer is 99.53%.
Claims (9)
1. the method preparing Fugu ocellatus toxin, it is characterised in that specifically include following steps:
Step one: soak
Ocellated puffer liver or roe are cut into inch strips or after block, soaks by deionized water or purified water,
To feed liquid, be dipped to feed liquid the most toxic till；
Step 2: sedimentation
The feed liquid of step one gained is placed in 6～10 DEG C of sedimentations place 10～12 hours, separates
Supernatant, and it is mixed to get clear liquor with supernatant again after centrifugal for precipitate also filter cleaner；
Step 3: flocculation
The clear liquor of step 2 gained ammonia spirit is adjusted pH to 7～9, adds flocculant molten
Liquid regulation pH to 4～6, stirs evenly, standing sedimentation, then separates supernatant, and by precipitate
Mix with supernatant again after high speed centrifugation filter cleaner；
Step 4: ion exchanges
Step 3 is mixed the feed liquid of gained, upper ion exchange column, exchanges to pH6.5～7.0,
Exchange rushes post extremely clarification by deionized water or purified water after terminating, then with acetum eluting,
Collect the component containing target product, adjust pH to 5～7 with ammonia spirit, be concentrated under reduced pressure to give dense
Contracting liquid；
Step 5: low pressure column chromatography
Concentrated solution and the dry silica gel of step 4 gained being mixed, upper Lower pressure silica gel chromatography column separates,
And add and consist of the eluent of n-butyl alcohol, acetic acid and water and carry out eluting, collect containing target product
Effluent part, be filtrated to get filtrate, be concentrated under reduced pressure to give concentrated solution；
Step 6: performance liquid chromatographic column
By the performance liquid chromatographic column after step 5 gained concentrated solution injection balance, with acetum
Eluting, collects the effluent concentrating under reduced pressure containing target product, obtains concentrated solution；
Step 7: crystallization
The concentrated solution of step 6 gained ammonia spirit is adjusted pH to 8～9, cold preservation, separates out knot
Crystalline substance, leaches, and with absolute ethanol washing, room temperature drying under reduced pressure i.e. obtains the crystallization of Fugu ocellatus toxin monomer.
The method preparing Fugu ocellatus toxin the most according to claim 1, it is characterised in that
In described step one, described ocellated puffer liver or roe be cut into 1～2cm bar or block,
Put and rustless steel soaking compartment add 1～the deionized water of 2 times or purified water are 6～10 DEG C of immersions,
And stir, after 18～24 hours, feed liquid is discarded to storage from the end opening of rustless steel soaking compartment. in good time
In hopper or settling tank.
The method preparing Fugu ocellatus toxin the most according to claim 2, it is characterised in that
In described step 3, it is that the clear liquor of step 2 gained is adjusted pH with the ammonia spirit of 25%
To 7～9, add flocculant solution to pH4～6, the speed with 50～300rpm/min stir 5～
10min, standing sedimentation 18～24 hours at temperature is 6～10 DEG C, after separating supernatant,
By precipitate through high speed centrifuge, centrifugal under rotating speed is 5000～10000rpm/min
20～40 minutes, and mix with supernatant again after filter cleaner.
The method preparing Fugu ocellatus toxin the most according to claim 3, it is characterised in that
Described flocculant is bodied ferric sulfate, polyaluminium sulfate, polyacrylamide or polyaluminium
Aluminum, injected volume is 0.01～5g/L.
The method preparing Fugu ocellatus toxin the most according to claim 4, it is characterised in that
In described step 4, be the feed liquid that step 3 is mixed gained, on rush through ammonification and with water
To the ion exchange column of pH8～9, exchange is to pH6.5～7.0, and wherein feed liquid and ion exchange
Amount ratio between resin is 40～60mL/g, and exchange is rushed by deionized water or purified water after terminating
Post is to clarification, then with 5～the acetum eluting of 10%, and acetum and upper feed liquid
Volume ratio is 1:90～180, collects the component containing target product, adjusts pH to 5～7 with ammonia,
At 0.9～1.0 atmospheric pressure concentrating under reduced pressure, concentrate 90～100 times, obtain concentrated solution.
The method preparing Fugu ocellatus toxin the most according to claim 5, it is characterised in that
The ion exchange resin used by ion exchange column in described step 4 is D152 weak-type
Macroporous acrylic cation exchange resin.
The method preparing Fugu ocellatus toxin the most according to claim 6, it is characterised in that
In described step 5, it is by the concentrated solution of step 4 gained, and dry silica gel is mixed, both
The ratio of amount be 0.8～1mL/g, upper Lower pressure silica gel chromatography column separates, and pressure is 0.7～0.9
Individual atmospheric pressure, addition consists of the eluent of n-butyl alcohol, acetic acid and water and carries out eluting, eluent
Being 50:1 with the volume ratio of upper feed liquid, wherein the volume ratio of n-butyl alcohol, acetic acid and water is respectively
For 5:3:1, collect the effluent part containing target product, with 0.45 μm filtering with microporous membrane,
Obtain filtrate with 0.9～1.0 atmospheric pressure, 90～100 times of concentrating under reduced pressure, obtain concentrated solution.
The method preparing Fugu ocellatus toxin the most according to claim 7, it is characterised in that
In described step 6, be by step 5 gained concentrated solution injection balance after high-efficient liquid phase color
Spectrum post, performance liquid chromatographic column is C18Post, applied sample amount is 0.2～0.4mL, with volume basis
Than being the acetum eluting of 0.4～0.6%, the volume ratio of eluent and upper feed liquid be 80～
150:1, monitors with differential refraction monitor, collect the effluent containing target product with 0.9～
1.0 atmospheric pressure, 90～100 times of concentrating under reduced pressure, obtain concentrated solution.
The method preparing Fugu ocellatus toxin the most according to claim 8, it is characterised in that
In described step 7, it is to be 8～10% by the concentrated solution volume fraction of step 6 gained
Ammonia adjust pH to 8～9, cold preservation, separate out crystallization, leach, with absolute ethanol washing,
-1.1～-0.9 atmospheric pressure under, normal temperature drying 24～within 48 hours, i.e. obtain Fugu ocellatus toxin monomer knot
Brilliant.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1385432A (en) * | 2001-05-15 | 2002-12-18 | 中国科学院水生生物研究所 | Method for purifying tetrodotoxin |
| WO2008102253A2 (en) * | 2007-02-23 | 2008-08-28 | The Hong Kong Polytechnic University | Method of biosynthesizing tetrodotoxin |
| CN101475575A (en) * | 2009-02-05 | 2009-07-08 | 王维国 | Extraction of tetraodontoxin, and preparation thereof used in drug dependence of dolantin |
| CN102584843A (en) * | 2011-01-18 | 2012-07-18 | 王秀菊 | Extraction method for high-purity tetrodotoxin |
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| CN1187356C (en) * | 2000-11-22 | 2005-02-02 | 南宁枫叶药业有限公司 | System for extracting tetradoxin with high yield |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1385432A (en) * | 2001-05-15 | 2002-12-18 | 中国科学院水生生物研究所 | Method for purifying tetrodotoxin |
| WO2008102253A2 (en) * | 2007-02-23 | 2008-08-28 | The Hong Kong Polytechnic University | Method of biosynthesizing tetrodotoxin |
| CN101475575A (en) * | 2009-02-05 | 2009-07-08 | 王维国 | Extraction of tetraodontoxin, and preparation thereof used in drug dependence of dolantin |
| CN102584843A (en) * | 2011-01-18 | 2012-07-18 | 王秀菊 | Extraction method for high-purity tetrodotoxin |
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