CN107917830A - A kind of method of extraction purification saxitoxin in class from toxiferous algae - Google Patents
A kind of method of extraction purification saxitoxin in class from toxiferous algae Download PDFInfo
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- G—PHYSICS
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Abstract
It is main to include extraction, extraction and three steps of column chromatography the present invention relates to a kind of method of extraction purification saxitoxin in class from toxiferous algae.Extracting solution includes methanol and chloroform, extractant includes n-hexane, chloroform, ethyl acetate and n-butanol, column chromatography includes just anti-phase silica gel column layer and high performance liquid chromatography, pass through optimization technological process, eluant, eluent, extracting solution and extractant are reasonably selected, each step is synergistic, finally obtains the saxitoxin of high-purity and high yield, yield reaches 4.58g, and purity reaches 99%.This method is succinctly efficient, saves cost, yield is higher, is conducive to industrial large-scale application.
Description
Technical field
The invention belongs to biological technical field, it is related to a kind of method of extraction purification saxitoxin in class from toxiferous algae.
Background technology
Ocean toxin is to pass to the life such as fish, shrimp and shellfish of algophagous by food chain by the toxic algae in ocean
Thing, and in its poisonous high-molecular compound that accumulation is formed in vivo.Ocean toxin species is various, wherein saxitoxin be harm compared with
One of big person.Saxitoxin includes paralytic shellfish poisoning (PSP), research of diarrhetic shellfish poisons, nerve saxitoxin and forgetful property shellfish
Toxin.Saxitoxin harm has sudden and popularity, has the characteristics that strong toxicity and Unpredictability, due to its toxicity
Greatly, reaction it is fast, without suitable antidote, bring many difficulties to prevention, saxitoxin is healthy and safe to consumer and coastal waters life
State brings serious threat, is ecocatas-trophe and great Marine Environmental Problems that international community pays close attention to jointly, therefore, saxitoxin
Research is of great significance.
At present, saxitoxin is also one of research, development progress field the most rapid, portion in marine biomaterial
Decibel toxoid has boundless answer in terms of the various medicines such as exploitation anticancer, town pain, anesthesia, antiviral, antibacterial
Use prospect.But since the density of the saxitoxin in natural environment is very low, its toxin is mainly by shellfish or fish
Enrichment of ingesting reaches a certain concentration and forms, thus, the source difficulties of toxin raw material dramatically constrain toxin into one
Step further investigation.At present, it is the separation toxiferous algae kind from natural surroundings to obtain one of approach of a large amount of saxitoxins, and is being tested
The large-scale culture that room is sheerly, therefrom extracts saxitoxin.
106119117 A of CN disclose a kind of preparation of the toxiferous algae and its standard sample of Azaspiracid toxin with answering
With extracting Azaspiracid in the shellfish positive after toxiferous algae, toxiferous algae nutrient solution or toxiferous algae of ingesting from culture
Toxin, obtains thick extracting toxin extracting solution：(2) by step (1) the thick extracting toxin extracting solution silicagel column and flash chromatography column into
Step (2) purified sample is crossed performance liquid chromatographic column by row purification (3), is freeze-dried, and white solid standard sample is made.
105237543 A of CN provide a kind of method for purifying and preparing the cruel base toxin of the cruel ammonia first of N- sulphurs.This method includes the training of toxiferous algae
The foster, extraction of toxin, the collection of toxin and purifying and etc..The bright one kind that is related to of 1629187 A of CN utilizes algae wet cell direct
The method of extraction and purification algae toxin.This method adds in the toxiferous algae frustule concentrate of certain volume under continuous stirring condition
Enter a certain amount of 100% methanol, adjust pH value to 2-7, be allowed to the 5-30 minutes broken frustules of supersound process of 40-400W
Release toxin completely, carries out separation of solid and liquid, the precipitation isolated is surpassed with a small amount of 50% methanol aqueous solution again on supercentrifuge
Sound extracts twice, merges the separating obtained supernatant of secondary centrifuging, and well directly evaporates or at 35-40 DEG C in 65-80 DEG C of water-bath
Under water bath condition using rotary evaporator evaporate, make cumulative volume reduce 50%, then add appropriate high-quality flocculant make it is residual
Remaining fine particle flocculation, and separation of solid and liquid is carried out on supercentrifuge, what is obtained upper asks liquid to cross 0.45 micron membrane filter, filtrate
The aqueous solution that can meet general algae toxin requirement of experiment as containing higher concentration algae toxin.
But the content of the saxitoxin obtained using current method for extraction and purification is relatively low, purity is not high, and step
Cumbersome, yield is barely satisfactory.Therefore, for saxitoxin extraction and purification process there are further Improvement requirement, this is exactly
Where power and starting point that the present invention is accomplished.
The content of the invention
In view of the deficiencies of the prior art with the demand in market, the present invention provides one kind from toxiferous algae class extraction purification shellfish
The method of toxoid, it is main to include extraction, extraction and three steps of column chromatography.This method is succinctly efficient, save cost, yield compared with
Height, is conducive to industrial large-scale application.
In a first aspect, the method that the present invention provides extraction purification saxitoxin in a kind of class from toxiferous algae, including following step
Suddenly：
(1) extract：Centrifuged after toxiferous algae class is expanded culture, chlorine is added after the frustule multigelation centrifuged
Imitative and methanol mixed liquid dipping, ultrasonication, reduction vaporization obtain total medicinal extract；
(2) extract：Deionized water dissolving is added to total medicinal extract, extraction is stood, and obtains extract；
(3) column chromatography：Extract is subjected to column chromatography, takes target fraction chromatographic isolation to obtain saxitoxin.
Inventor is on the basis of fully research saxitoxin and extraction process, by many experiments optimization technological process,
Eluant, eluent, extracting solution and extractant are reasonably selected for the physicochemical property of saxitoxin, each step is synergistic, finally improves shellfish
The yield and purity of toxoid.
Preferably, the additive amount of the mixed liquor of step (1) chloroform and methanol is 3-6 times of frustule volume, such as
It can be 3 times, 4 times, 5 times or 6 times, be preferably 5 times.
Preferably, the volume ratio (1-10) of the chloroform and methanol:1, such as can be 1:1,2:1,3:Isosorbide-5-Nitrae:1,5:1,
6:1,7:1,8:1,9:1 or 10:1, it is preferably 1:1.
Preferably, the condition of step (1) described centrifugation is 2000-8000rpm, for example, can be 2000 rpm,
3000rpm, 4000rpm, 5000rpm, 6000rpm, 7000rpm or 8000rpm, are preferably 5000rpm；
Preferably, the number of freezing and thawing is 1-5 times, such as can be 1 time, 2 times, 3 times, 4 times or 5 times, is preferably 2 times.
Preferably, the soaking time is 20-60min, for example, can be 20min, 30min, 40 min, 50min or
60min, is preferably 30min.
Preferably, the intensity of the ultrasonication is 10-30%, such as can be 10%, 20% or 30%, is preferably
20%.
Preferably, the additive amount of step (2) described deionized water is 2-4 times of total medicinal extract volume, for example, can be 2 times, 3
Times or 4 times, be preferably 3 times.
Preferably, step (2) extraction is extracted using extractant.
Preferably, the additive amount of the extractant is 2-4 times of total medicinal extract volume, such as can be 2 times, 3 times or 4 times,
Preferably 3 times.
Preferably, the extractant is any one in n-hexane, chloroform, ethyl acetate or n-butanol or at least two
Combination, such as can be the combination of n-hexane, chloroform and ethyl acetate, chloroform, the combination of ethyl acetate and n-butanol or just
The combination of hexane, chloroform, ethyl acetate and n-butanol, is preferably n-hexane, chloroform, ethyl acetate and n-butanol.
Preferably, the extraction specifically comprises the following steps：After adding the first extractant into total medicinal extract of dissolving, fill
Stood after point mixing, be extracted to color it is clear it is shallow after change one kind again, sequentially add n-hexane, chloroform, ethyl acetate by this step
And n-butanol, finally obtain n-hexane, chloroform, ethyl acetate and n-butyl alcohol extract.
Preferably, the time of repose is 10-30min, such as can be 10min, 20min or 30min, is preferably
20min。
Preferably, step (3) described column chromatography is normal-phase silica gel column chromatography, reversed-phase silica gel column chromatography, ion exchange column layer
Any of analysis or adsorpting column chromatography or at least two combination, such as can be normal-phase silica gel column chromatography and reverse phase silica gel column
The combination of the combination of chromatography, ion-exchange chromatography and adsorpting column chromatography, is preferably normal-phase silica gel column chromatography and reverse phase silica gel column
The combination of chromatography.
Preferably, the purification on normal-phase silica gel eluent described in step (3) includes petroleum ether, ethyl acetate, n-hexane, chloroform, third
In ketone or methanol any one or at least two combination, such as can be the combination of petroleum ether, ethyl acetate, n-hexane,
Chloroform, the combination or ethyl acetate of acetone and methanol, the combination of n-hexane and chloroform, are preferably ethyl acetate, n-hexane and chlorine
Imitative combination.
Preferably, the reverse phase silica gel eluent described in step (3) includes the mixed liquor of water, n-butanol, acetonitrile and methanol.
Preferably, the chromatographic isolation described in step (3) is affinity chromatography, electrochromatophoresis, gas-chromatography or high-efficient liquid phase color
Any of spectrum separation or at least two combination, such as can be the combination of affinity chromatography and electrochromatophoresis, electrochromatophoresis
It is preferably high performance liquid chromatography with the combination of gas-chromatography.
Preferably, the eluent of the high performance liquid chromatography is acetonitrile and the mixed liquor of methanol.
Preferably, the volume ratio of the acetonitrile and methanol is 9:1.
The method of extraction purification saxitoxin, includes the following steps in a kind of class from toxiferous algae：
(1) extract：Toxiferous algae class is expanded and is centrifuged after cultivating with the rotating speed of 2000-8000rpm, the algae centrifuged
After cell freeze thawing 1-5 times, chloroform of 3-6 times equivalent to frustule volume and the mixed liquid dipping 20-60min of methanol, chlorine are added
Imitative and methanol volume ratio is (1-10):1, then using intensity as 10%-30% sonicated cells, be evaporated under reduced pressure obtain it is total
Medicinal extract；
(2) extract：Dissolved to the addition of total medicinal extract equivalent to 2-4 times of deionized water of total medicinal extract volume, using total
10-30min is stood after 2-4 times of the extractant extraction of medicinal extract volume, finally obtains extract；
Extraction specifically comprises the following steps：After adding the first extractant into total medicinal extract of dissolving, it is sufficiently mixed rear quiet
Put, be extracted to color it is clear it is shallow after change one kind again, sequentially add n-hexane, chloroform, ethyl acetate and n-butanol by this step, most
After obtain n-hexane, chloroform, ethyl acetate and n-butyl alcohol extract；
(3) column chromatography：Extract is subjected to normal-phase silica gel column chromatography and reversed-phase silica gel column chromatography, purification on normal-phase silica gel elution respectively
Liquid includes the combination of any one or at least two in petroleum ether, ethyl acetate, n-hexane, chloroform, acetone or methanol, anti-phase
Silica gel eluent includes the mixed liquor of water, n-butanol, acetonitrile and methanol；
Target fraction is taken to carry out high performance liquid chromatography, the eluent of the high performance liquid chromatography is that volume ratio is 9:1 second
Saxitoxin is prepared in the mixed liquor of nitrile and methanol, chromatographic isolation.
Second aspect, the present invention provide a kind of saxitoxin of method extraction purification according to first aspect.
Compared with prior art, beneficial effects of the present invention：
Method for extraction and purification provided by the present invention is carried by the optimization of the frustule extraction purification condition to toxiferous algae class
The reasonably optimizing of the conditions such as liquid, extractant, eluent is taken, not only increases the yield and purity of saxitoxin in toxiferous algae class,
Yield is up to 4.58g, and purity can reach 99%, and extraction purification step is simple, and cost is low, saves manpower thing
Power, is conducive to industrial large-scale application.
Embodiment
Further to illustrate the technological means and its effect of the invention taken, below in conjunction with being preferable to carry out for the present invention
Example further illustrates technical scheme, but the present invention is not limited in scope of embodiments.
Embodiment 1
The method of extraction purification saxitoxin, includes the following steps in a kind of class from toxiferous algae：
(1) extract：Toxiferous algae class is added in 2L natural sea-water nutrient solutions according to weight in wet base 50g, cultivate 48h after with
The rotating speed of 5000rpm is centrifuged, and after the frustule freeze thawing centrifuged 2 times, adds chloroform of 5 times equivalent to frustule volume
With the mixed liquid dipping 30min of methanol, the volume ratio of chloroform and methanol is 1:1, it is then thin as 20% ultrasonication using intensity
Born of the same parents, reduction vaporization obtain total medicinal extract；
(2) extract：Dissolved to the addition of total medicinal extract equivalent to 3 times of deionized water of total medicinal extract volume, using total leaching
3 times of extractant of paste volume, after adding the first extractant, 20min is stood after being sufficiently mixed, be extracted to color it is clear it is shallow after
Change one kind again, n-hexane, chloroform, ethyl acetate and n-butanol sequentially added by this step, finally obtain n-hexane, chloroform,
Ethyl acetate and n-butyl alcohol extract；
(3) column chromatography：Extract is subjected to normal-phase silica gel column chromatography and reversed-phase silica gel column chromatography, purification on normal-phase silica gel elution respectively
Liquid is the mixed liquor of ethyl acetate, n-hexane and chloroform, and reverse phase silica gel eluent is the mixing of water, n-butanol, acetonitrile and methanol
Liquid.Target fraction is taken to carry out high performance liquid chromatography, eluent is that volume ratio is 9:1 acetonitrile and the mixed liquor of methanol, collect mesh
Chromatographic peak is marked, freeze-drying, is made white solid.
Embodiment 2
The method of extraction purification saxitoxin, includes the following steps in a kind of class from toxiferous algae：
(1) extract：Toxiferous algae class is added in 2L natural sea-water nutrient solutions according to weight in wet base 50g, cultivate 48h after with
The rotating speed of 2000rpm is centrifuged, and after the frustule freeze thawing centrifuged 1 time, adds chloroform of 3 times equivalent to frustule volume
With the mixed liquid dipping 30min of methanol, the volume ratio of chloroform and methanol is 10:1, then using intensity as 10% ultrasonication
Cell, reduction vaporization obtain total medicinal extract；
(2) extract：Dissolved to the addition of total medicinal extract equivalent to 2 times of deionized water of total medicinal extract volume, using total leaching
2 times of extractant of paste volume, after adding the first extractant, 20min is stood after being sufficiently mixed, be extracted to color it is clear it is shallow after
Change one kind again, n-hexane, chloroform, ethyl acetate and n-butanol sequentially added by this step, finally obtain n-hexane, chloroform,
Ethyl acetate and n-butyl alcohol extract；
(3) column chromatography：Extract is subjected to normal-phase silica gel column chromatography and reversed-phase silica gel column chromatography, purification on normal-phase silica gel elution respectively
Liquid is the mixed liquor of ethyl acetate, n-hexane and chloroform, and reverse phase silica gel eluent is the mixing of water, n-butanol, acetonitrile and methanol
Liquid.Target fraction is taken to carry out high performance liquid chromatography, eluent is that volume ratio is 9:1 acetonitrile and the mixed liquor of methanol, collect mesh
Chromatographic peak is marked, freeze-drying, is made white solid.
Embodiment 3
The method of extraction purification saxitoxin, includes the following steps in a kind of class from toxiferous algae：
(1) extract：Toxiferous algae class is added in 2L natural sea-water nutrient solutions according to weight in wet base 50g, cultivate 48h after with
The rotating speed of 6000rpm is centrifuged, and after the frustule freeze thawing centrifuged 3 times, adds chloroform of 4 times equivalent to frustule volume
With the mixed liquid dipping 20min of methanol, the volume ratio of chloroform and methanol is 8:1, it is then thin as 15% ultrasonication using intensity
Born of the same parents, reduction vaporization obtain total medicinal extract；
(2) extract：Dissolved to the addition of total medicinal extract equivalent to 2 times of deionized water of total medicinal extract volume, using total leaching
4 times of extractant of paste volume, after adding the first extractant, 20min is stood after being sufficiently mixed, be extracted to color it is clear it is shallow after
Change one kind again, n-hexane, chloroform, ethyl acetate and n-butanol sequentially added by this step, finally obtain n-hexane, chloroform,
Ethyl acetate and n-butyl alcohol extract；
(3) column chromatography：Extract is subjected to normal-phase silica gel column chromatography and reversed-phase silica gel column chromatography, purification on normal-phase silica gel elution respectively
Liquid is the mixed liquor of ethyl acetate, n-hexane and chloroform, and reverse phase silica gel eluent is the mixing of water, n-butanol, acetonitrile and methanol
Liquid.Target fraction is taken to carry out high performance liquid chromatography, eluent is that volume ratio is 9:1 acetonitrile and the mixed liquor of methanol, collect mesh
Chromatographic peak is marked, freeze-drying, is made white solid.
Embodiment 4
The method of extraction purification saxitoxin, includes the following steps in a kind of class from toxiferous algae：
(1) extract：Toxiferous algae class is added in 2L natural sea-water nutrient solutions according to weight in wet base 50g, cultivate 48h after with
The rotating speed of 5000rpm is centrifuged, and after the frustule freeze thawing centrifuged 2 times, adds chloroform of 5 times equivalent to frustule volume
With the mixed liquid dipping 30min of methanol, the volume ratio of chloroform and methanol is 1:1, it is then thin as 20% ultrasonication using intensity
Born of the same parents, reduction vaporization obtain total medicinal extract；
(2) extract：Dissolved to the addition of total medicinal extract equivalent to 3 times of deionized water of total medicinal extract volume, using total leaching
3 times of extractant of paste volume, after adding the first extractant, 20min is stood after being sufficiently mixed, be extracted to color it is clear it is shallow after
One kind is changed again, chloroform, ethyl acetate and n-butanol are sequentially added by this step, finally obtains chloroform, ethyl acetate and positive fourth
Alcohol extract；
(3) column chromatography：Extract is subjected to positive and reversed-phase silica gel column chromatography respectively, purification on normal-phase silica gel eluent is n-hexane
With the mixed liquor of chloroform, reverse phase silica gel eluent is the mixed liquor of water, n-butanol, acetonitrile and methanol.Target fraction is taken to carry out high
Effect liquid phase chromatogram, eluent are that volume ratio is 9:1 acetonitrile and the mixed liquor of methanol, collect target chromatographic peak, are freeze-dried, system
Obtain white solid.
Embodiment 5
The method of extraction purification saxitoxin, includes the following steps in a kind of class from toxiferous algae：
(1) extract：Toxiferous algae class is added in 2L natural sea-water nutrient solutions according to weight in wet base 50g, cultivate 48h after with
The rotating speed of 7000rpm is centrifuged, and after the frustule freeze thawing centrifuged 3 times, adds chloroform of 4 times equivalent to frustule volume
With the mixed liquid dipping 50min of methanol, the volume ratio of chloroform and methanol is 6:1, it is then thin as 25% ultrasonication using intensity
Born of the same parents, reduction vaporization obtain total medicinal extract；
(2) extract：Dissolved to the addition of total medicinal extract equivalent to 3 times of deionized water of total medicinal extract volume, using total leaching
3 times of extractant of paste volume, after adding the first extractant, 20min is stood after being sufficiently mixed, be extracted to color it is clear it is shallow after
One kind is changed again, n-hexane, chloroform and n-butanol are sequentially added by this step, finally obtains n-hexane, chloroform and n-butanol extraction
Take thing；
(3) column chromatography：Extract is subjected to normal-phase silica gel column chromatography and reversed-phase silica gel column chromatography, purification on normal-phase silica gel elution respectively
Liquid is n-hexane and the mixed liquor of chloroform, and reverse phase silica gel eluent is the mixed liquor of water, n-butanol, acetonitrile and methanol.Take target
Fraction carries out high performance liquid chromatography, and eluent is that volume ratio is 9:1 acetonitrile and the mixed liquor of methanol, collect target chromatographic peak,
Freeze-drying, is made white solid.
Comparative example 1
Compared with Example 1, except step (1) described mixed liquor is methanol, addition volume is 1 times of frustule volume
Outside, other conditions are same as Example 1.
Comparative example 2
Compared with Example 1, except step (2) described extractant is methanol and ethyl acetate, additive amount is total medicinal extract body
Long-pending 1 times is outer, and other conditions are same as Example 1.
Comparative example 3
Compared with Example 1, except the eluent of the normal-phase silica gel column chromatography of step (3) is water, reversed-phase silica gel column chromatography
Eluent for outside ethanol and chloroform, other conditions are same as Example 1.
Sample test
The white solid that embodiment and comparative example obtain is weighed, by mass spectrum and nuclear-magnetism identification molecular weight and spectrum, knot
Fruit is as shown in table 1：
Table 1
As shown in Table 1, embodiment 1-5 can effectively extract yield and the higher saxitoxin of purity, its molecular weight, light
Spectrum is consistent with scallop toxin standard items.The wherein yield highest of embodiment 1, reaches 4.58g, and purity reaches 99%.And comparative example
For 1-3 after extracting solution, extractant and eluant composition and additive amount is changed, comparative example 1 can not extract saxitoxin, contrast
The yield and purity of example 2-3 is extremely low.
In conclusion method for extraction and purification provided by the present invention passes through the frustule extraction purification condition to toxiferous algae class
Optimization, such as extracting solution, extractant, the reasonably optimizing of eluent condition, greatly improve saxitoxin in toxiferous algae class
Yield and purity, yield are up to 4.58g, and purity reaches 99%.And extraction purification step is simple, cost is low, saves people
Power material resources, are conducive to industrial large-scale application.
Applicant states that the present invention illustrates the method detailed of the present invention, but not office of the invention by above-described embodiment
It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., all fall within protection scope of the present invention and the open scope.
Claims (10)
1. a kind of method of extraction purification saxitoxin in class from toxiferous algae, it is characterised in that include the following steps：
(1) extract：Will toxiferous algae class expand culture after be centrifuged, after the frustule multigelation centrifuged add chloroform and
The mixed liquid dipping of methanol, ultrasonication, reduction vaporization obtain total medicinal extract；
(2) extract：Deionized water dissolving is added to total medicinal extract, extraction is stood, and obtains extract；
(3) column chromatography：Extract is subjected to column chromatography, takes target fraction chromatographic isolation to obtain saxitoxin.
2. the according to the method described in claim 1, it is characterized in that, addition of the mixed liquor of step (1) chloroform and methanol
Measure as 3-6 times of frustule volume, be preferably 5 times；
Preferably, the volume ratio (1-10) of the chloroform and methanol:1, it is preferably 1:1.
3. method according to claim 1 or 2, it is characterised in that the condition of step (1) described centrifugation is 2000-
8000rpm, is preferably 5000rpm；
Preferably, the number of freezing and thawing is 1-5 times, is preferably 2 times；
Preferably, the soaking time is 20-60min, is preferably 30min；
Preferably, the intensity of the ultrasonication is 10-30%, is preferably 20%.
4. method according to any one of claim 1-3, it is characterised in that the addition of step (2) described deionized water
Measure as 2-4 times of total medicinal extract volume, be preferably 3 times；
Preferably, step (2) extraction is extracted using extractant；
Preferably, the additive amount of the extractant is 2-4 times of total medicinal extract volume, is preferably 3 times；
Preferably, the extractant is any one in n-hexane, chloroform, ethyl acetate or n-butanol or at least two group
Close, be preferably n-hexane, chloroform, ethyl acetate and n-butanol.
5. according to the described method of any one of claim 1-4, it is characterised in that the extraction specifically comprises the following steps：
Stood after the mixing of the first extractant is added into total medicinal extract of dissolving, add a kind of lower extractant mixing and stand, add successively
Extract is finally obtained after entering all extractants；
Preferably, the time of repose is 10-30min, is preferably 20min.
6. according to the method any one of claim 1-5, it is characterised in that step (3) described column chromatography is positive silicon
In plastic column chromatography, reversed-phase silica gel column chromatography, ion-exchange chromatography or adsorpting column chromatography any one or at least two group
Close, be preferably the combination of normal-phase silica gel column chromatography and reversed-phase silica gel column chromatography.
7. according to the method any one of claim 1-6, it is characterised in that the purification on normal-phase silica gel elution described in step (3)
Liquid includes the mixed liquor of any one or at least two in petroleum ether, ethyl acetate, n-hexane, chloroform, acetone or methanol, excellent
Elect the mixed liquor of ethyl acetate, n-hexane and chloroform as；
Preferably, the reverse phase silica gel eluent described in step (3) includes the mixed liquor of water, n-butanol, acetonitrile and methanol.
8. according to the described method of any one of claim 1-7, it is characterised in that the chromatographic isolation described in step (3) is parent
It is preferably height with any of chromatography, electrochromatophoresis, gas-chromatography or high performance liquid chromatography separation or at least two combination
Effect liquid phase chromatogram；
Preferably, the eluent of the high performance liquid chromatography is acetonitrile and the mixed liquor of methanol；
Preferably, the volume ratio of the acetonitrile and methanol is 9:1.
9. according to the method any one of claim 1-8, it is characterised in that include the following steps：
(1) extract：Toxiferous algae class is expanded and is centrifuged after cultivating with the rotating speed of 2000-8000rpm, the frustule centrifuged
After freeze thawing 1-5 times, add chloroform of 3-6 times equivalent to frustule volume and the mixed liquid dipping 20-60min of methanol, chloroform and
The volume ratio of methanol is (1-10):1, then using intensity as 10-30% sonicated cells, be evaporated under reduced pressure obtain total medicinal extract；
(2) extract：Dissolved to the addition of total medicinal extract equivalent to 2-4 times of deionized water of total medicinal extract volume, using total medicinal extract
2-4 times of extractant of volume, after adding the first extractant, stands 10-30min after being sufficiently mixed, it is shallow clearly to be extracted to color
Change one kind again afterwards, sequentially add the extractant by this step, finally obtain extract；
Extractant is the combination of any one or at least two in n-hexane, chloroform, ethyl acetate or n-butanol.
(3) column chromatography：Extract is subjected to normal-phase silica gel column chromatography and reversed-phase silica gel column chromatography, purification on normal-phase silica gel eluent bag respectively
Include in petroleum ether, ethyl acetate, n-hexane, chloroform, acetone or methanol any one or at least two combination, reverse phase silica gel
Eluent includes the mixed liquor of water, n-butanol, acetonitrile and methanol；
Target fraction is taken to carry out high performance liquid chromatography, the eluent of the high performance liquid chromatography is that volume ratio is 9:1 acetonitrile and
Saxitoxin is prepared in the mixed liquor of methanol, chromatographic isolation.
A kind of 10. saxitoxin of method extraction purification according to any one of claim 1-9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711036004.5A CN107917830A (en) | 2017-10-30 | 2017-10-30 | A kind of method of extraction purification saxitoxin in class from toxiferous algae |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201711036004.5A CN107917830A (en) | 2017-10-30 | 2017-10-30 | A kind of method of extraction purification saxitoxin in class from toxiferous algae |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103163001A (en) * | 2013-03-27 | 2013-06-19 | 云南省环境监测中心站 | Method for extracting and purifying microcystic toxins LR and RR by taking cyanobacterial bloom in Dian Lake as raw material |
| CN105198900A (en) * | 2015-09-22 | 2015-12-30 | 国家海洋环境检测中心 | Pure yessotoxin (YTX) extracting and preparing method |
| CN106119117A (en) * | 2016-06-12 | 2016-11-16 | 中国水产科学研究院黄海水产研究所 | The toxiferous algae of a kind of Azaspiracid toxin and the preparation and application of standard sample thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103163001A (en) * | 2013-03-27 | 2013-06-19 | 云南省环境监测中心站 | Method for extracting and purifying microcystic toxins LR and RR by taking cyanobacterial bloom in Dian Lake as raw material |
| CN105198900A (en) * | 2015-09-22 | 2015-12-30 | 国家海洋环境检测中心 | Pure yessotoxin (YTX) extracting and preparing method |
| CN106119117A (en) * | 2016-06-12 | 2016-11-16 | 中国水产科学研究院黄海水产研究所 | The toxiferous algae of a kind of Azaspiracid toxin and the preparation and application of standard sample thereof |
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Application publication date: 20180417 |