CN1245406C - Method for extracting tetrodotoxin from globefish livers - Google Patents

Abstract

The present invention relates to a method for extracting tetrodotoxin from the liver of a blowfish, which is characterized in that the liver of a blowfish is cut up, soaked at low temperature, stirred, filtered and frozen, and then is repeatedly soaked for many times to obtain crude toxic liquid; after the crude toxic liquid is frozen and placed to obtain precipitation substances; after the precipitation substances are heated to be boiled, the toxic liquid is discharged; after the toxic liquid is refrozen, supernatant toxic liquid is separated, and the pH value of the supernatant toxic liquid is regulated; after activated carbon is added to the supernatant toxic liquid, the mixture is stirred and filtered; cation 110 resin is mixed with the clear liquid, and the mixture is stirred and filtered; the mixture passes through the cation 110 resin, and is eluted by acid after being saturated, and the eluted part is collected; the pH value is regulated; after cooling and standing still to obtain crystals, mother liquid is sucked out and filtered; then the pH valve is regulated again, and the mother liquid is frozen and crystallized, and stands still; the pH is repeatedly regulated for many times, white powdery crystals are obtained. By using the present invention, the recovery rate of the tetrodotoxin can be furthest increased, impurities can be effectively removed, and the extraction time can be shortened. Thus, the loss of the biological activity of the tetrodotoxin is reduced to a minimum margin. In the processes of extraction and separation, the treatment is carried out at low temperature, and thus, raw materials are easy to obtain.

Description

Extract the method for tetraodotoxin from the globe fish liver

Technical field

The present invention relates to a kind of method of extracting the high purity tetraodotoxin from the globe fish liver.

Background technology

Tetraodotoxin (TTX) claims former globefish element (Spheroidine), globefish toxin (Fugupoison) etc. again.TTX is a kind of alkaloids toxin of separating in the globe fish body the earliest.The pharmacy monumental work Compendium of Material Medica of China Ming Dynasty has been done comparatively detailed introduction to filefish toxicity, TTX is one of human the most malicious known non-protein matter so far, serious symptom can appear in patient that tetraodotoxin is poisoned immediately, and threat to life, mortality ratio higher (being generally 60%).But TTX has important application value, except can be used as medicine, also rehabilitate addicts, there is tempting application prospect aspect such as anti-arrhythmia, respiration inhibition, prevention renal failure, cardiovascular, analgesia, toponarcosis.TTX is the white crystalline solid, needle-like-rhombus, and odorless, tasteless, easy moisture absorption deliquescence, molecular formula is C ₁₁H ₁₇N ₃O ₈, molecular weight 319.27.1 carbocyclic ring, 1 guanidine radicals are arranged in the structure of TTX, 6 hydroxyls, the ring that separates that has a semialdehyde sugar lactone being connected in C-5 and C-10 position, in alkali aqueous solution, easily decompose, for example in 5% (mass percentage concentration) potassium hydroxide solution, under 90～100 ℃ temperature, can resolve into yellow crystal 2-amino-methylol-8-hydroxyl-quinazoline.The used raw material of existing extracting method be red fin east pure (Fugu rubripes), purple Fugu (F.porphyre us), leopard line Fugu (F, pardalis) and the ovary of back of the body spot Fugu (F.stictonotos).This method mainly comprises with clear water cleans ovary, mashes, and 3 hours after-filtration of hot-water soak should precipitates after the filtering also concentrated with acidifying with acetic acid; Then, in spissated filtrate, add plumbic acetate, produce new precipitation; After this is precipitated filtering once more, in filtrate, add ammoniacal liquor, obtain containing the lead precipitation of toxin; Use H ₂S takes off lead, and once more filtrate decompression is concentrated; At last, residue is dissolved in acetic acid the part insolubles can occur, with its filtering, adds ether in filtrate, will be the thick toxin that Tian Yuanfa makes after the precipitation drying that produce.Also there are human phospho-wolframic acid, picric acid mercury, phenylhydrazine, picric acid mercury and picrolonic acid etc. to handle ovary, the thick toxin LD that obtains successively ₅₀Be 800 μ g/kg.This method more complicated, and the thick content of toxins that makes is very low.But this thick toxin can not crystallization, uses Al afterwards ₂O ₃Column chromatography can be from this thick toxin (LD ₅₀, obtain pure product crystallization in 10mg/kg).It is that 5.5 centimetres circular filter paper carries out chromatography that Jin Tianhehecun uses diameter, and shared 500 filter paper pile column, and the 50th to 70 has flooded 500mg toxicity is the tetraodotoxin aqueous solution of 200 μ g/kg, and pile is with phenol-capable wash-out waterborne of 90%; Use ether flush away phenol again, wash through cold water lixiviate, concentrating under reduced pressure, ether again, drying obtains 200mg toxin (LD ₅₀, 120 μ g/kg).Handle through ethanol, acetic acid washing and ether repeatedly again, obtain 10mg crystallization tetraodotoxin (LD ₅₀, 10 μ g/kg).Jin Tianyuhecun tests tetraodotoxin with active carbon method again.From the ovary of Fugu rubripes (Temmincket Schlegel), purple Fugu and leopard line Fugu of purchase, obtain tetraodotoxin crystallization (LD by this method ₅₀, 10-13 μ g/kg).Use this method, can obtain the 10g tetraodotoxin from the 1000kg ovary, the rate of recovery is very low.

Summary of the invention

The purpose of this invention is to provide a kind of method from globe fish liver extraction tetraodotoxin, it can remedy

The above-mentioned deficiency of prior art.

A kind of method from globe fish liver extraction tetraodotoxin earlier with the ocellated puffer liver chopping, with 0-15 ℃ of immersion of deionized water, is stirred, and the vat liquor filtration is freezing, repeats to soak for several times again, after vat liquor is nontoxic, obtains crude venom; This crude venom precipitation occurs after freezing placement, the sucking-off supernatant liquid is heated to after the boiling, and venom is emitted, freezing again after, tell the supernatant venom, obtain orange-yellow clarification crude venom after filtering; Regulate the pH value, add gac, stir, filter, obtain light yellow clarifying liquid; It is characterized in that Hydrogen positively charged ion 110 resins are mixed with this clear liquor, stir, filter, the little yellow clear liquor that obtains, through ammonium type positively charged ion 110 resins, the wash-out part is collected in the pickling deresination of saturated back; Regulate the pH value, cooling is left standstill, and occurs crystal after a couple of days, with the mother liquor sucking-off, filters, and transfers pH again, and is freezing, leaves standstill, and crystallization is regulated pH for several times repeatedly, gets the white powder crystal; Or wash-out partly repeated absorption with macroporous adsorbent resin D101, the back obtains venom with alcohol, aqueous acid wash-out, regulates the pH value and obtains the tetraodotoxin crystal.

The part by weight of described deionized water and liver is 1-5: 1 described Heating temperature is more than 100 ℃, and heat-up time is more than 2 minutes.Described adjusting crude venom pH is to 3-7, and the proportional range of the ml number of venom and the g number of gac is 10-50: 1.

Advantage of the present invention is that (1) repeats the rate of recovery that sorptive power improves tetraodotoxin to greatest extent to venom; (2) adopt the h type resin material to remove impurity effectively, reduced extraction time, the biological activity loss of tetraodotoxin is minimized; (3) in the extraction separation process, keep lesser temps to handle; (4) from the extraction raw material of tetraodotoxin, liver is than the easier acquisition of ovary.

Embodiment

Embodiment

1, from icebox, takes out 25 kilograms of ocellated puffer livers, shred with slicing machine, put into volume and be 80 liters polyethylene drum, add 50 liters of deionized waters, low temperature (0 ℃-15 ℃) soaked 20 hours, stirred in immersion process in good time, and speed is slower during stirring, to next day vat liquor is emitted from the bucket end opening, relief liquor filters through 350 order yarn thin,tough silk.This is a toxin vat liquor for the first time, and it is standby that this liquid is squeezed into freezing 4 hours of freezer by peristaltic pump.Repeat again to soak 3 times, obtain 185 liters of crude venoms.The 5th time vat liquor stops to soak after the detection of mouse method is nontoxic.

2, above-mentioned crude venom after placing, icebox flocks occurred, with peristaltic pump sucking-off supernatant liquid, squeeze in the heating kettle, the bottom precipitated liquid also adds in the pot after with filter paper filtering, be heated to the boiling and kept 10 minutes after, venom is emitted, and it is freezing to return icebox after the cooling, leaves standstill to treat that it goes out contamination precipitation, after freezing 2 hours, get the supernatant venom in the bucket, obtain orange-yellow clarification crude venom after filtering, adjust pH to 4.0, the gac that adds treated mistake, venom (ml) is 50: 1 with gac (g) ratio, stirs 30 minutes, obtains light yellow clarifying liquid after filtration.

3, will transform good Hydrogen 110 resins and add light yellow clarifying liquid, resin and venom volume ratio 1: 15, quiet phase reaction was stirred 30 minutes, obtained little yellow clear liquor after filtration.This little yellow clear liquor is through ammonia type 110 resins, and flow velocity is 50ml/min.With mouse method monitoring saturated conditions, stop material loading after saturated.

4, the HAC elutriant of preparation 15% (concentration expressed in percentage by weight) with its flow velocity wash-out venom with 10ml/min, is collected the wash-out venom with iodine flask.Regulate pH value to 9.5, put into refrigerator, leave standstill, crystal occurs after a couple of days.With the mother liquor sucking-off in the iodine flask, with the attached wall thing of certain density acetate dissolution, filter with sand core funnel, transfer pH to 9.5 again, put into refrigerator, leave standstill crystallization.4 final white powder crystal 111.8mg that get are 87% through the quantitative purity of HPLC so repeatedly.

If 5 aforesaid operations do not obtain crystallization, then do following processing: it is 7.5 that the venom that obtains in the elution process is regulated the pH value with weak ammonia, occurs flocks in the regulate process, filters, the D101 resin is added the good clear liquid of filtration, quiet phase reaction is stirred, after about 2 hours in good time, the mixed solution of D101 resin and venom of taking a morsel filters, the mouse method detects the virulence of venom, and abdominal injection 0.5ml does not think extremely promptly that filtrate is nontoxic, and the back filters out the D101 resin with B.

6, use deionized water at 250ml polyethylene centrifugal barrel wash-in D101 resin, the ratio of this resin and water is 1: 3, stirs evenly the D101 resin with glass stick, stop after 15 minutes stirring, freezing, use centrifugal 10 minutes of whizzer, abandoning supernatant adds washing again, repeats washed several times with water.With washed D101 resin wet method dress post,, collect elutriant, direct crystallization with the mixed solution wash-out of ethanol, acetic acid, water (their weight ratio is 20: 2: 78).Obtain tetraodotoxin crystal 5 4.25mg, quantitative through HPLC, purity is 87%.

Claims (4)

1, a kind of method from globe fish liver extraction tetraodotoxin earlier with the ocellated puffer liver chopping, with 0-15 ℃ of immersion of deionized water, is stirred, and the vat liquor filtration is freezing, repeats to soak for several times again, after vat liquor is nontoxic, obtains crude venom; This crude venom precipitation occurs after freezing placement, the sucking-off supernatant liquid is heated to after the boiling, and venom is emitted, freezing again after, tell the supernatant venom, obtain orange-yellow clarification crude venom after filtering; Regulate the pH value, add gac, stir, filter, obtain light yellow clarifying liquid; It is characterized in that Hydrogen positively charged ion 110 resins are mixed with this clear liquor, stir, filter, the little yellow clear liquor that obtains, through ammonium type positively charged ion 110 resins, the wash-out part is collected in the pickling deresination of saturated back; Regulate the pH value, cooling is left standstill, and occurs crystal after a couple of days, with the mother liquor sucking-off, filters, and transfers pH again, and is freezing, leaves standstill, and crystallization is regulated pH for several times repeatedly, gets the white powder crystal; Or wash-out partly repeated absorption with macroporous adsorbent resin D101, the back obtains venom with alcohol, aqueous acid wash-out, regulates the pH value and obtains the tetraodotoxin crystal.

2, the method for claim 1, the part by weight that it is characterized in that described deionized water and liver is 1-5: 1.

3, the method for claim 1 is characterized in that described Heating temperature is more than 100 ℃, and heat-up time is more than 2 minutes.

4, the method for claim 1 is characterized in that described adjusting crude venom pH to 3-7, and the proportional range of the ml number of venom and the g number of gac is 10-50: 1.