CN1706836A - Process of separating fucoxanthin from algae - Google Patents
Process of separating fucoxanthin from algae Download PDFInfo
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- CN1706836A CN1706836A CN 200410020727 CN200410020727A CN1706836A CN 1706836 A CN1706836 A CN 1706836A CN 200410020727 CN200410020727 CN 200410020727 CN 200410020727 A CN200410020727 A CN 200410020727A CN 1706836 A CN1706836 A CN 1706836A
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Abstract
The present invention relates to the extraction and separation of fucoxanthin, and is especially the process of separating fucoxanthin from algae. The process of separating fucoxanthin from algae includes the following steps: cleaning fresh algae or defrozen algae to eliminate surface salt ion, eliminating surface water, leaching algae with dimethyl sulfoxide in 2-6 ml for each gram of algae in dark for 15-60 min, and final extracting coloring matter from dimethyl sulfoxide leached liquid with the mixture solution of ethyl acetate and ammonium sulfate. The present invention has the advantages of simple operation, high separation speed and high yield, and is suitable for industrial production.
Description
Technical field
The present invention relates to the extraction separation of fucoxanthin, is a kind of method of separating fucoxanthin from marine alga specifically.
Background technology
Fucoxanthin (fucoxanthin, this paper is abbreviated as fucox) be fucoxanthin again, is tawny, is one of important member of carotenoid family, has very strong antioxygenation.Molecular formula is C
40H
60O
6Structural formula is:
Fucoxanthin mainly exists in brown alga and the diatom, catches photopigment as periphery, and the content in frond is very big.As far back as the sixties in 20th century, people keep fowls with alga food with regard to discovery can deepen the color of yolk, and this effect is because poultry has absorbed the fucoxanthin in the frond and caused.Japanese in recent years scholar finds that fucoxanthin has very strong anti-tumor activity in doing antineoplastic experimental study.Murakami studies show that fucoxanthin was a kind of effective inhibitors of Mammals replicative DNA polysaccharase (pol α) in 2002.The refined history leader's in the thin river of Hokkaido University research group found in 2003, pigment composition in the marine alga such as wakame and sea-tangle reduces the protein of overslaugh cancer cells natural death in the cancer cells in a large number, impel rectum cancer cell natural death and anticancer propagation, the appreciation rate of cancer cells reduces 15% approximately.Experiment proves also that simultaneously the effect of fucoxanthin anticancer propagation is considerably beyond the fruits and vegetables pigment composition.
Fucoxanthin is a kind of natural active matter, because it has strong reducing property, is again the compound of provitamin A class simultaneously, therefore has very important potential value of exploiting and utilizing.Can be used as: (1) medicine, as be used for oncotherapy, anti-ageing medicine, cosmetics additive and prevent and treat ophthalmic diseases etc.; (2) food, drink additive; (3) fishery products bait improves immunizing power, increases color and luster; (4) poultry and livestock feed is enriched in the ovum, so not only makes darkening of yolk, and the nutritive value of egg also significantly improves.
The extensive extractive technique of fucoxanthin and the research of application thereof are not also carried out at home, domesticly do not see that as yet the patent of relevant fucoxanthin delivers.
Sea-tangle is important large-scale economical alga, the first place that its breed scale and output account for cultivating seaweed, and world's annual production is about 3,100,000 tons, and China accounts for over half.The sea-tangle of China is to be introduced into DaLian, China by Japan at first, and the sea-tangle cultivation industry has been developed in the back, successfully overcome the difficulty of aestivating after, now carry out widely by north to south in that China is coastal.Sea-tangle contains abundant fucoxanthin, and sea-tangle output is big in addition, and is therefore with the material of sea-tangle as extensive extraction fucoxanthin, very convenient effective, can not have the insufficient problem in source.But sea-tangle contains a large amount of brown alga colloid and polysaccharide simultaneously, has brought very big difficulty for the separation and purification work of fucoxanthin.
The conventional method of extracting pigment from fresh kelp or plant leaf is: earlier big fresh frond is cut into small pieces, the acetone and other organic solvent that adds certain volume is again ground, and the pigment that extracts and the mixture of residue are with which floor filtered through gauze or centrifugation.This kind method is used for from the defective below the existence of brown algas such as sea-tangle extraction fucoxanthin:
(1) waste time and energy, extractive process will be ground, therefore big frond must cut very broken, and pigment and residue will pass through centrifugal or filtered through gauze.
(2) owing to phycocolloid and polysaccharide content in the kelps such as sea-tangle are big especially, bring very big difficulty for extraction work.
(3) yield is low, uses the acetone extracting, simultaneously with chlorophyll a [chl a] and a large amount of having extracted of Chlorofucsin [chl c].
Summary of the invention
The object of the present invention is to provide a kind of simple to operate, speed is fast, yield is high separates fucoxanthin from marine alga method.
The objective of the invention is to be achieved through the following technical solutions:
A kind of method of from marine alga, separating fucoxanthin, its specific operation process is: with the marine alga of fresh marine alga or stored frozen after room temperature is thawed, clean 3-5 time with seawater, removing surface impurity and colloid cleans 2-3 time with distilled water again, remove the salt ion on frond surface, dry up then, dry in the shade or remove its surface-moisture with thieving paper; Again with marine alga with dimethyl sulfoxide (DMSO) (DMSO) lixiviate 15～60 minutes in the dark, the consumption of dimethyl sulfoxide (DMSO) is the every gram of a 2～6ml/ marine alga; Final refining get final product fucoxanthin.
Described marine alga is sea-tangle, sargassun (Sargassum), black wrack (Fucus), chickweed (Endarachne), capsule algae (Colpnmenina), rope algae (Chorda), wakame (Undaria), bulk kelp (Macrocystis), siliquosa Pelvetia (Pelvetia), sargassum kjellmanianum Yendo (S.kjellmanianum), Sargassum fusiforme (S.fusiforme) or Sargassum (S.pallidumm) etc.; In the leaching process, when extraction time is 40～50 minutes, when the consumption of DMSO is the every gram of 4～6ml/ marine alga, separating effect the best, the yield of fucoxanthin is the highest.
Pigment can carry out purifying according to the following procedure in the vat liquor of the present invention:
The removal of DMSO: because the boiling point too high (189 ℃) of DMSO, unsuitable evaporative removal during low temperature, and the too high structure that can destroy fucoxanthin of vaporization temperature, so colorant mixture is difficult to be further purified and obtains dry powder formulations.Pure DMSO is dissolved in ethyl acetate, and when being mixed with the aqueous solution, DMSO then is extracted to water, separate with ethyl acetate, and DMSO (easily produces precipitation at sulfate of ammoniac solution in the concentration 〉=0.5mol/l).(concentration is respectively 0.25mol/l, 0.5mol/l, 1mol/l, 2mol/l, 3mol/l) extraction, very fast layering (see figure 7) to add 1 times of volumes of acetic acid ethyl ester and 1 times of volume sulfate of ammoniac among the present invention.The upper strata safran is an ethyl acetate layer, and most of pigment all forwards the upper strata to.As seen from Figure 7, when not adding sulfate of ammoniac or sulfate of ammoniac concentration too hour, layering is not obvious, and lower floor's (sulfate of ammoniac and DMSO mixolimnion) is also contained than polychrom; It is better that concentration is separated effect during for 0.5mol/l, but lower floor still has small amount of coloring matter, use ethyl acetate extraction 2-3 time again after, lower floor's pigment disappears substantially; Separate during for 1mol/l effectively when concentration, lower floor does not almost have pigment to distribute, and (2-3mol/l) was little to the separating effect influence when concentration was high again.Upper solution can add 0.2 times of volume 0.5mol/l sulfate of ammoniac solution extraction and (further remove the DMSO of small amount of residual) several times.
The separation and purification of pigment: can utilize conventional silica gel column chromatography, but concrete implementation step reference literature ' Vitamin A-related compounds, all-trans retinal and retinoic acids, selectively inhibit activities of mammalian replicative DNA polymerases ', Chikako Murakami etc.Biochimica et Biophysica Acta, 2002 (1574): 85-92.Can obtain the very high fucoxanthin of purity through 3-4 silica gel column chromatography.
The present invention has following advantage:
1. simple to operate.Operating procedure of the present invention is very simple, good separating effect, used lixiviate reagent and plant and instrument all seldom, so expense is few, cost is very low.
2. velocity of separation is fast.The isolating time of the present invention is shorter, and leaching process only needs dozens of minutes to get final product.
3. yield height.The present invention adopts dimethyl sulfoxide (DMSO) to extract fucoxanthin from Radix Laminariae, adopts the extracting method of acetone solvent to compare the yield height with prior art, purity is good, and the agents useful for same methyl sulfoxide is a kind of good medical solvent, and toxicological harmless can not influence the native conformation of fucoxanthin.
4. lay a good foundation for batch production production.The present invention proposes a kind of fucoxanthin extracting method of simple and effective, and preliminary study multiple factor to the influence that fucoxanthin extracts, help the further research of its extraction process, thereby finally be used for the scale operation of batch production.
Description of drawings
Fig. 1 for Radix Laminariae with acetone (the acetone consumption is the every gram Radix Laminariae of 3ml/) respectively behind lixiviate 30min (1) and the 70min (2), the visible absorption spectrum of vat liquor.
Fig. 2 for Radix Laminariae with acetone (the acetone consumption is the every gram Radix Laminariae of 3ml/) respectively behind lixiviate 30min (1) and the 70min (2), the normal temperature fluorescence emission spectrum (excitation wavelength EM=452nm) of vat liquor.
Fig. 3 D be Radix Laminariae with DMSO (the DMSO consumption is respectively 2ml, 4ml, the every gram Radix Laminariae of 6ml/) lixiviate 40min after, the visible absorption spectrum of vat liquor.
Fig. 3 A is a Radix Laminariae with DMSO (the DMSO consumption is respectively 2ml, 4ml, the every gram Radix Laminariae of 6ml/) lixiviate 40min, and residue uses acetone (the acetone consumption is the every gram Radix Laminariae of 3ml/) respectively behind the lixiviate 30min again, the visible absorption spectrum of residue vat liquor.
Fig. 4 D be Radix Laminariae with DMSO (the DMSO consumption is respectively 2ml, 4ml, the every gram Radix Laminariae of 6ml/) lixiviate 40min after, the normal temperature fluorescence emission spectrum (excitation wavelength EM=452nm) of vat liquor.
Fig. 4 A is a Radix Laminariae with DMSO (the DMSO consumption is respectively 2ml, 4ml, the every gram Radix Laminariae of 6ml/) lixiviate 40min, after residue is used acetone (the acetone consumption is the every gram Radix Laminariae of 3ml/) difference lixiviate 30min again, the normal temperature fluorescence emission spectrum (excitation wavelength EM=452nm) of residue vat liquor.
Fig. 5 D is after Radix Laminariae is distinguished lixiviate 20,40,60min with DMSO (the DMSO consumption is the every gram Radix Laminariae of 4ml/), the visible absorption spectrum of vat liquor.
Fig. 5 A is that Radix Laminariae is distinguished lixiviate 20,40,60min with DMSO (the DMSO consumption is the every gram Radix Laminariae of 4ml/), after residue is used acetone (the acetone consumption is the every gram Radix Laminariae of 3ml/) difference lixiviate 30min again, and the visible absorption spectrum of residue vat liquor.
Fig. 6 D is after Radix Laminariae is distinguished lixiviate 20,40,60min with DMSO (the DMSO consumption is the every gram Radix Laminariae of 4ml/), the normal temperature fluorescence emission spectrum (excitation wavelength EM=452nm) of vat liquor.
Fig. 6 A is that Radix Laminariae is distinguished lixiviate 20,40,60min with DMSO (the DMSO consumption is the every gram Radix Laminariae of 4ml/), after residue is used acetone (the acetone consumption is the every gram Radix Laminariae of 3ml/) difference lixiviate 30min again, the normal temperature fluorescence emission spectrum (excitation wavelength EM=452nm) of residue vat liquor.
Fig. 7 uses the sulfate of ammoniac solution extraction result of ethyl acetate and different concns mutually for DMSO; Wherein sulfate of ammoniac concentration is respectively: 1. 3mol/l; 2. 2mol/l; 3. 1mol/l; 4. 0.5mol/l; 5. 0.25mol/l,
Embodiment
Sea-tangle rhizoid, sargassun and black wrack with cleaning 3-5 time with seawater respectively, after removal surface impurity and the part colloid, are directly used in and extract pigment or place-20 ℃ of refrigerators freezing standby.
Embodiment 1
Fresh or freezing Radix Laminariae is taken out, after room temperature is thawed, clean 3 times, remove the salt ion on algae surface, blot surface-moisture with thieving paper then with distilled water.Take by weighing the 1g Radix Laminariae and put into small beaker, add DMSO 3ml, seal the beaker mouth with two-layer preservative film, lixiviate 50min pours vat liquor in the brown bottle in the dark, again with pouring in the brown bottle behind the 3ml distilled water cleaning residue.Measure absorbance A
665, A
631, A
582, A
480, calculate pigment concentration by following formula: [chl a]=A
665/ 72.8; [chl c]=(A
631+ A
582-0.298 A
665)/61.8; [Fucoxanthin]=(A
480-0.723 (A
631+ A
582-0.298 A
665)-0.049 A
665)/130.Calculating yield is foundation with the amount of pigment that proposes in every gram Radix Laminariae, the results are shown in Table 1.
The contained amount of pigment and the ratio of the shared total pigment of fucoxanthin in the every gram Radix Laminariae of table 1:
| Handle | Fucox(μg) | Fucox/chla+chlc |
| DMSO?50min | 64.79 | 1.52 |
Embodiment 2
Fresh or freezing Radix Laminariae is taken out, after room temperature is thawed, clean 3 times, remove the salt ion on algae surface, blot surface-moisture with thieving paper then with distilled water.Take by weighing the 1g Radix Laminariae and put into small beaker, add DMSO 4ml, seal the beaker mouth, behind the lixiviate 15min, vat liquor is poured in the brown bottle in the dark, again with pouring in the brown bottle behind the 4ml distilled water cleaning residue with two-layer preservative film.Absorbance measurement and pigment concentration method of calculation are with embodiment 1, and calculating yield is foundation with the amount of pigment that proposes in every gram Radix Laminariae, the results are shown in Table 2.
The contained amount of pigment and the ratio of the shared total pigment of fucoxanthin in the every gram Radix Laminariae of table 2:
| Handle | Fucox(μg) | Fucox/chla+chlc |
| DMSO?15min | 18.9 | 1.07 |
Embodiment 3
Fresh or freezing Radix Laminariae is taken out, after room temperature is thawed, clean 5 times, remove the salt ion on algae surface, blot surface-moisture with thieving paper then with distilled water.Take by weighing the 1g Radix Laminariae and put into small beaker, add DMSO 7ml, seal the beaker mouth, behind the lixiviate 30min, vat liquor is poured in the brown bottle in the dark, again with pouring in the brown bottle behind the 7ml distilled water cleaning residue with two-layer preservative film.Absorbance measurement and pigment concentration method of calculation are with embodiment 1, and calculating yield is foundation with the amount of pigment that proposes in every gram Radix Laminariae, the results are shown in Table 3.
The contained amount of pigment and the ratio of the shared total pigment of fucoxanthin in the every gram Radix Laminariae of table 3:
| Handle | Fucox(μg) | Fucox/chla+chlc |
| DMSO?30min | 93.87 | 0.52 |
Embodiment 4
Fresh or freezing sargassun root is taken out, after room temperature is thawed, clean 3 times, remove the salt ion on algae surface, blot surface-moisture with thieving paper then with distilled water.Take by weighing the 1g sargassun and put into small beaker, add DMSO 3ml, seal the beaker mouth, behind the lixiviate 50min, vat liquor is poured in the brown bottle in the dark, again with pouring in the brown bottle behind the 3ml distilled water cleaning residue with two-layer preservative film.Absorbance measurement and pigment concentration method of calculation are with embodiment 1, and calculating yield is foundation with the amount of pigment that proposes in every gram sargassun, the results are shown in Table 4.
The every amount of pigment contained in the sargassun and the ratio of the shared total pigment of fucoxanthin of restraining of table 4:
| Handle | Fucox(μg) | Fucox/chla+chlc |
| DMSO?50min | 79.15 | 0.56 |
Embodiment 5
Fresh or freezing black wrack root is taken out, after room temperature is thawed, clean 3 times, remove the salt ion on algae surface, blot surface-moisture with thieving paper then with distilled water.Take by weighing the 1g black wrack and put into small beaker, add DMSO 3ml, seal the beaker mouth, behind the lixiviate 50min, vat liquor is poured in the brown bottle in the dark, again with pouring in the brown bottle behind the 3ml distilled water cleaning residue with two-layer preservative film.Absorbance measurement and pigment concentration method of calculation are with embodiment 1, and calculating yield is foundation with the amount of pigment that proposes in every gram black wrack, the results are shown in Table 5.
The every amount of pigment contained in the black wrack and the ratio of the shared total pigment of fucoxanthin of restraining of table 5:
| Handle | ?Fucox(μg) | ?Fucox/chla+chlc |
| ?DMSO?50min | ?42.13 | ?1.56 |
Embodiment 6 (Comparative Examples)
Fresh or freezing Radix Laminariae is taken out, after room temperature is thawed, clean 3 times, remove the salt ion on frond surface, blot surface-moisture with thieving paper then with distilled water.Take by weighing two groups of 0.9g Radix Laminariaes, be placed in 2 small beakers, press the every gram Radix Laminariae of acetone 3ml/, add 2.7ml100% acetone respectively, seal the beaker mouth, respectively behind lixiviate 30min and the 70min, vat liquor is poured in the brown bottle in the dark with two-layer preservative film.Measure its visible absorption spectrum (see figure 1) and fluorescence emission spectrum (see figure 2).With 100% acetone is blank, measures absorbance A
661, A
628, A
580, A
470, calculate pigment concentration by following formula: [chl a]=A
661/ 83.3; [chl c]=(A
628+ A
580-0.239 A
661)/62.0; [Fucoxanthin]=(A
470-0.774 (A
628+ A
580-0.239 A
661)-0.031 A
661)/142, calculating yield is foundation with the amount of pigment that proposes in every gram Radix Laminariae, the results are shown in Table 1.
The contained amount of pigment and the ratio of the shared total pigment of fucoxanthin in the every gram Radix Laminariae of table 6:
| Handle | ?Chl?a(μg) | ?Chl?c(μg) | ?Fucox(μg) | ?Fucox/chla+chlc |
| Acetone 30min | ?245.88 | ?38.19 | ?106.70 | ?0.38 |
| Acetone 70min | ?310.19 | ?51.89 | ?127.11 | ?0.35 |
Find out by Fig. 1: after Radix Laminariae is distinguished lixiviate 30min (1) and 70min (2) with acetone, both spectral shapes are similar, maximum absorption band is 432nm, secondly be 661nm, charateristic avsorption band for chlorophyll a, the small peak at 620nm and 580nm place is the absorption peak of Chlorofucsin, does not see the charateristic avsorption band of fucoxanthin thus among the figure.
Find out by Fig. 2: after Radix Laminariae is distinguished lixiviate 30min (1) and 70min (2) with acetone, excitation wavelength EM=452nm, both spectral shapes are similar, the maximum fluorescence emission peak is about 646.5nm, secondly be about 676.8nm, the former is the fluorescence emission peak of fucoxanthin, and the latter is the feature emission peak of chlorophyll a.As can be seen, wherein the fluorescence ratio of chlorophyll a contribution is very big.
Embodiment 7 (Comparative Examples)
Fresh or freezing Radix Laminariae is taken out, after room temperature is thawed, clean 3 times, remove the salt ion on algae surface, blot surface-moisture with thieving paper then with distilled water.Take by weighing 2.55g, 3.2g, 3.0g Radix Laminariae respectively, be placed in 3 small beakers, by the every gram Radix Laminariae of DMSO/ is that 2ml, 4ml, 6ml add DMSO 5.1ml, 12.8ml respectively, (numbering is followed successively by 1D to 18ml, 2D, 3D), seal the beaker mouth, in the dark lixiviate 40min with two-layer preservative film, after the end extract is poured in the brown bottle, poured in the brown bottle after cleaning residue with 1.25ml, 3.2ml, 4.5ml distilled water respectively again.Residue blots with thieving paper after cleaning 3 times with distilled water, presses then described in the embodiment 6, add respectively 7.65ml, 9.6ml, 9ml acetone (numbering is followed successively by 1A, 2A, 3A), lixiviate 30min is filled into vat liquor in the brown bottle respectively.Measure its visible absorption spectrum (see Fig. 3 A, 3D) and fluorescence emission spectrum (see Fig. 4 A, 4D).Pigment content is measured, and DMSO presses described in the embodiment 1 mutually, and acetone is pressed described in the embodiment 6 mutually, and calculating yield is foundation with the amount of pigment that proposes in every gram Radix Laminariae, the results are shown in Table 7.
The contained amount of pigment and the ratio of the shared total pigment of fucoxanthin in the every gram Radix Laminariae of table 7:
| Handle | Chla(μg) | Chlc(μg) | Fucox(μg) | Fucox/chla+chlc |
| ?1D | ??4.13 | ??19.13 | ??11.25 | ??0.48 |
| ?2D | ??24.75 | ??42 | ??65.63 | ??0.98 |
| ?3D | ??41.63 | ??30.38 | ??90.38 | ??1.26 |
| ?1A | ??134.07 | ??--- | ??57.83 | ??0.43 |
| ?2A | ??185.25 | ??18.32 | ??31.28 | ??0.15 |
| ?3A | ??164.82 | ??22.7 | ??34.26 | ??0.18 |
Found out by Fig. 3 D: maximum absorption band is 452nm, from fucoxanthin; 2D and 3D have little absorption peak at 581nm, 630nm and 665nm place, and wherein 581nm and 630nm are from Chlorofucsin, and 665nm is from chlorophyll a; 1D does not almost have absorption peak at this 3 place, but the 452nm absorption peak of 1D is also low than 2D and 3D.
Found out by Fig. 3 A: maximum absorption band is 431nm, is 661nm secondly, and there is an acromion at the 417nm place, and 3 derive from chlorophyll a; The little absorption peak of 578nm and 617nm derives from chl c, does not have the charateristic avsorption band of fucoxanthin.
Found out by Fig. 4 D: the maximum fluorescence emission peak is 654-655nm, is the characteristic fluorescence emission peak of fucoxanthin, does not derive from the characteristic fluorescence emission peak of chlorophyll a.
Found out by Fig. 4 A: the maximum fluorescence emission peak is 674-679nm, is the characteristic fluorescence emission peak of chlorophyll a, and wherein the 645nm small peak of the 644nm of 1A and 3A derives from fucoxanthin, and the fucoxanthin fluorescent emission acromion of 2A is 647nm.
Embodiment 8
Fresh or freezing Radix Laminariae is taken out, after room temperature is thawed, clean 3 times, remove the salt ion on algae surface, blot surface-moisture with thieving paper then with distilled water.Take by weighing 2.0g, 3.2g, 2.0g Radix Laminariae respectively, be placed in 3 small beakers, by the every gram Radix Laminariae of DMSO/ is that 4ml adds DMSO 8ml, 12.8ml, 8ml respectively, seal the beaker mouth with two-layer preservative film, respectively lixiviate 20,40 in the dark, 60min (numbering is followed successively by 4D, 5D, 6D), after the end vat liquor is poured in the brown bottle, poured in the brown bottle after cleaning residue with 2ml, 3.2ml, 2ml distilled water respectively again.Residue blots with thieving paper after cleaning 3 times with distilled water, then by embodiment 6 add respectively 6ml, 9.6ml, 6ml acetone (be numbered 4A, 5A, 6A), lixiviate 30min is filled into extract in the brown bottle respectively.Measure its visible absorption spectrum (see Fig. 5 A, D) and fluorescence emission spectrum (see Fig. 6 A, D).Pigment content is measured, and DMSO presses described in the embodiment 1 mutually, and acetone is pressed described in the embodiment 6 mutually, and calculating yield is foundation with the amount of pigment that proposes in every gram Radix Laminariae, the results are shown in Table 8.
The contained amount of pigment and the ratio of the shared total pigment of fucoxanthin in the every gram Radix Laminariae of table 8:
| Handle | Chla(μg) | Chlc(μg) | Fucox(μg) | Fucox/chla+chlc |
| ?4D | ??26.25 | ??34.5 | ??55.5 | ??0.91 |
| ?5D | ??24.75 | ??42 | ??65.63 | ??0.98 |
| ?6D | ??48 | ??61.5 | ??77.63 | ??0.71 |
| ?4A | ??119.28 | ??--- | ??31.58 | ??0.35 |
| ?5A | ??185.25 | ??18.32 | ??31.28 | ??0.15 |
| ?6A | ??195.87 | ??30.8 | ??43.26 | ??0.19 |
Found out by Fig. 5 D: maximum absorption band is 452nm, from fucoxanthin; 5D and 6D have little absorption peak at the 665nm place, from chlorophyll a; 4D has little absorption peak at 581nm and 630nm place, from Chlorofucsin.
Found out by Fig. 5 A: maximum absorption band is 431nm, is 661nm secondly, and there is an acromion at the 416nm place, and 3 derive from chlorophyll a; The little absorption peak of 578nm and 617nm derives from chlc, does not have the charateristic avsorption band of fucoxanthin.
Found out by Fig. 6 D: the maximum fluorescence emission peak is 654-655nm, is the characteristic fluorescence emission peak of fucoxanthin, does not derive from the characteristic fluorescence emission peak of chlorophyll a.
Found out by Fig. 6 A: the maximum fluorescence emission peak is that 676-679nm is the characteristic fluorescence emission peak of chlorophyll a, and wherein the 645nm small peak of the 644.8nm of 4A and 6A derives from fucoxanthin, and the fucoxanthin fluorescent emission acromion of 5A is 647nm.
Embodiment 9
The removal of DMSO: because the boiling point too high (189 ℃) of DMSO, unsuitable evaporative removal during low temperature, and the too high structure that can destroy fucoxanthin of vaporization temperature, so colorant mixture is difficult to be further purified and obtains dry powder formulations.Pure DMSO is dissolved in ethyl acetate, and when being mixed with the aqueous solution, DMSO then is extracted to water, separate with ethyl acetate, and DMSO (easily produces precipitation at sulfate of ammoniac solution in the concentration 〉=0.5mol/l).
The present invention will be by the DMSO vat liquor of embodiment 7 and embodiment 8 gained, and (concentration is respectively 0.25mol/l, 0.5mol/l, 1mol/l, 2mol/l, 3mol/l) extraction, very fast layering (see figure 7) to add 1 times of volumes of acetic acid ethyl ester and 1 times of volume sulfate of ammoniac respectively.The upper strata is an ethyl acetate layer, presents safran, and most of pigment all is extracted to ethyl acetate layer.As seen from Figure 7, when not adding sulfate of ammoniac or sulfate of ammoniac strength of solution too hour, layering is not obvious, and lower floor's (sulfate of ammoniac and DMSO mixolimnion) is also contained than polychrom; It is better that concentration is separated effect during for 0.5mol/l, but lower floor still has small amount of coloring matter, use ethyl acetate extraction 2-3 time again after, lower floor's pigment disappears substantially; Separate during for 1mol/l effectively when concentration, lower floor does not almost have pigment to distribute, and (2-3mol/l) was little to the separating effect influence when concentration was high again.Upper solution can add 0.2 times of volume 0.5mol/l sulfate of ammoniac solution extraction (with the DMSO of further removal small amount of residual) several times.
Dimethyl sulfoxide (DMSO) (DMSO), molecular formula are (CH
3)
2SO, it is a kind of aprotic polar solvent, toxicity is extremely low.Chemical reaction is had special solvent effect to DMSO and to the dissolution characteristics of many materials, " Dimethyl Sulfoxide " is otherwise known as.Be widely used in separation and purification, the arviculture breeding of refining of petroleum, organic synthesis, synthon, agricultural chemicals, medicine and gas, liquid mixture, the fields such as selection by mutation of microorganism.In medicine intermediate is synthetic, use a lot in the medicine industry field as reaction solvent, it has anti-inflammatory, pain relieving, diuresis, calmness, stimulate circulation and wound healing and the effect of obvious suppression tumour is arranged, human body is had very strong penetrating power and to the carrying of other drug, synergism, can increase drug absorption and improve curative effect.
The present invention is a solvent with DMSO, has founded the experimental technique of the higher fucoxanthin of from the kelp sea-tangle easy dna purity rapidly and efficiently.Experimental result shows:
(1) can be illustrated by picture: the effect of using acetone extraction is well below the effect of handling with DMSO, during with acetone treatment with a large amount of having extracted of chlorophyll a, the absorption peak (Fig. 1) that does not at all have fucoxanthin in its visible absorption spectrum, fluorescence emission spectrum excites with 452nm, and the fluorescence intensity of chlorophyll a and fucoxanthin is (Fig. 2) quite.And all select DMSO to handle, and the absorption peak of its visible absorption spectrum fucoxanthin is very strong, and the absorption peak of chla and chlc very weak (Fig. 3 D, 5D), in the fluorescence emission spectrogram, have only fucoxanthin the characteristic fluorescence peak (Fig. 4 D, 6D).
(2) by the table explanation, carry out different treatment with DMSO:
A. different volumes (2 times, 4 times, 6 times) is handled the identical time (40min), increase along with the DMSO treatment capacity, the fucoxanthin yield improves constantly (6 times of volumes are handled: every gram Radix Laminariae contains 90.38 μ g), and purity is maximum (1.26) also, secondly are 4 times of processing (purity is 0.98); Residue with acetone treatment after, its fucoxanthin yield of the residue of 4 times of processing is minimum, purity is also minimum.Detailed data sees Table 2.
B. handle different times (20,40,60min) with 4 times of volume DMSO, the yield that 40min handles is the highest, and purity is best.Detailed data sees Table 3.
(3) this experiment is by extracting fucoxanthin with DMSO from freezing Radix Laminariae, the yield height, and purity is good, and method is simply quick, and cost is low, and DMSO is an innoxious solvent.By the multiplefactor screening, find with 3-6 times of volume (DMSO: algae, ml:g) extracting 40-50min best results.The present invention has not only proposed a kind of fucoxanthin extracting method of simple and effective, and preliminary study multiple factor to the influence that fucoxanthin extracts, will help the further research of its extraction process, thereby finally be used for the scale operation of batch production.
Claims (7)
1. method of from marine alga, separating fucoxanthin, it is characterized in that: specific operation process is, with fresh marine alga or freezing marine alga after thawing under the room temperature, clean with distilled water, remove its surface-moisture then, again with marine alga with dimethyl sulfoxide (DMSO) lixiviate in the dark, extraction time is 15～60 minutes, the consumption of dimethyl sulfoxide (DMSO) is the every gram of a 2～6ml/ marine alga.
2. press the described method of separating fucoxanthin from marine alga of claim 1, it is characterized in that: described freezing marine alga can be cleaned the back in below-5 ℃ or-5 ℃ by with fresh marine alga with seawater, freezing acquisition more than 2 hours.
3. by the described method of separating fucoxanthin from marine alga of claim 1, it is characterized in that: described marine alga is sea-tangle, sargassun, black wrack, chickweed, capsule algae, rope algae, wakame, bulk kelp, siliquosa Pelvetia, sargassum kjellmanianum Yendo, Sargassum fusiforme or Sargassum.
4. by the described method of separating fucoxanthin from marine alga of claim 1, it is characterized in that: described extraction time is 40～50 minutes, and the consumption of dimethyl sulfoxide (DMSO) is the every gram of a 4～6ml/ marine alga.
5. by the described method of separating fucoxanthin from marine alga of claim 1, it is characterized in that: the removal of dimethyl sulfoxide (DMSO) in the vat liquor after the lixiviate adds 0.5-1.5 times of volumes of acetic acid ethyl ester and 1 times of volume sulfate of ammoniac solution extraction in vat liquor; Pigment is transferred in the ethyl acetate layer on upper strata.
6. by the described method of separating fucoxanthin from marine alga of claim 5, it is characterized in that: described sulfate of ammoniac concentration is 0.25-3mol/l.
7. by the described method of separating fucoxanthin from marine alga of claim 5, it is characterized in that: the separation and purification of pigment, the ethyl acetate layer liquid that will contain pigment can obtain the very high fucoxanthin of purity through silica gel column chromatography.
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Cited By (7)
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| CN101914075A (en) * | 2010-08-23 | 2010-12-15 | 秦皇岛领先科技发展有限公司 | Method for extracting fucoxanthin from brown algae |
| WO2011047530A1 (en) * | 2009-10-20 | 2011-04-28 | 北京绿色金可生物技术股份有限公司 | Carotenoid derivatives, preparation method and use thereof |
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| CN103965143A (en) * | 2013-01-30 | 2014-08-06 | 中国科学院海洋研究所 | Method for extracting and separating fucoxanthin from marine unicellular diatom |
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| CN101914075A (en) * | 2010-08-23 | 2010-12-15 | 秦皇岛领先科技发展有限公司 | Method for extracting fucoxanthin from brown algae |
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| CN102336725B (en) * | 2011-06-10 | 2014-09-10 | 秦皇岛大惠生物技术有限公司 | Method for extracting fucoxanthine-containing concentrate, product obtained through method, and application of product |
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| CN103725034B (en) * | 2013-12-24 | 2015-05-27 | 上海海洋大学 | Method for extracting pigment from Sargassum horneri |
| CN103833692A (en) * | 2014-02-21 | 2014-06-04 | 宁波大学 | Method for extracting high-purity fucoxanthin from seaweeds |
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| CN105820142A (en) * | 2016-04-29 | 2016-08-03 | 宁波大学 | Pretreatment method for brown alga sample for fucoxanthin extraction |
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