CN103012327B - Preparation method of fucoxanthin - Google Patents
Preparation method of fucoxanthin Download PDFInfo
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- CN103012327B CN103012327B CN201210564339.5A CN201210564339A CN103012327B CN 103012327 B CN103012327 B CN 103012327B CN 201210564339 A CN201210564339 A CN 201210564339A CN 103012327 B CN103012327 B CN 103012327B
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- 238000002360 preparation method Methods 0.000 title claims abstract description 38
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- AQLRNQCFQNNMJA-UHFFFAOYSA-N fucoxanthin Natural products CC(=O)OC1CC(C)(C)C(=C=CC(=CC=CC(=CC=CC=C(/C)C=CC=C(/C)C(=O)CC23OC2(C)CC(O)CC3(C)C)C)CO)C(C)(O)C1 AQLRNQCFQNNMJA-UHFFFAOYSA-N 0.000 title abstract description 14
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- 239000012141 concentrate Substances 0.000 claims description 16
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- 229920005989 resin Polymers 0.000 claims description 7
- 241000593522 Sargassum thunbergii Species 0.000 claims description 6
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- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 claims description 5
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- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 description 4
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- Epoxy Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a preparation method of fucoxanthin and relates to the fucoxanthin. The preparation method of the fucoxanthin comprises the steps that a cleaned brown algae material is aired, and soaked by an organic solvent for extraction; an obtained extracting solution is condensed, and a fucoxanthin crude extract is obtained; the fucoxanthin crude extract is subjected to column chromatography separation; a fraction of the fucoxanthin is collected and condensed, and a fucoxanthin raw material is obtained; the obtained fucoxanthin raw material is dissolved by the organic solvent and transferred to a sample introduction bottle of a semipreparative/preparative HPLC (high performance liquid chromatograph); the semipreparative/preparative HPLC is started for separation and purification; fucoxanthin purification liquid is collected automatically through ultraviolet on-line detection or by a fraction collector triggered by a mass spectrum signal; and after the collected fucoxanthin purification liquid is decompressed, condensed, frozen and dried, the high-purity fucoxanthin is obtained. Through testing, the purity of the prepared fucoxanthin is greater than 99%.
Description
Technical field
The present invention relates to fucoxanthine, especially relate to the preparation method that a kind of purity is greater than high-purity fucoxanthine of 99%.
Background technology
Fucoxanthine (fucoxanthin) is also known as brown alga flavine, in edible brown alga, as wakame (Alariaceae, Undariapinnatifida) natural carotenoid, extracted in sea-tangle (Laminaria japonica Aresch), have at the two ends of its rigidity alltrans long-chain that chemical property is active respectively 5,6-epoxy unsaturation propadiene bond structure, because of and differ from other carotenoid molecules, there is very strong biological activity.In recent years, its various biological activity is proved, and among some potential activity are also actively being sought by scientists, oneself becomes one of main attack focus of current marine drug research and development at present.
Fucoxanthine has the multiple biological activitys such as antitumor, anti-inflammatory, anti-oxidant, fat-reducing.First, at anti-tumor aspect, the reported first such as nineteen ninety Okuzumi, can reduce the propagation of the Ct Characteristics of Nasopharyngeal Carcinoma Involving The Pterygopatatine cell strain (GOTO) of 62% after fucoxanthine 10 μ g/mL hatches 3 days.Okuztllni etc. study confirmation, and fucoxanthine can suppress to urge the cancer thing tetradecane phthalein Buddhist ripple acetic acid mouse skin ornithine that extremely (TPA) induces by strong skin and take off the enhancing of shuttle enzymic activity, infers that it may have restraining effect to skin carcinoma accordingly.The reports such as okuzumi in 1993, the duodenal cancer of fucoxanthine to N-ethyl-N '-nitro-nitroso-guanidine induction is formed inhibited.Masashi etc. report the effect of fucoxanthine to acute myeloid leukaemia HL-60 cell strain, and result display fucoxanthine can play significant inhibited proliferation to HL-60 cell.Elichi etc. study discovery, and fucoxanthine can obviously reduce prostate cancer cell survival rate, and cell death inducing.The people such as Swadesh K study and find that the growth of fucoxanthine to people liver cancer HePG2 cell is inhibited, and therefore, fucoxanthine has restraining effect in various degree to kinds of tumor cells.Secondly, research finds that fucoxanthine also has an antioxidation activity in vitro, and its anti-oxidant activity is even better than vitamins C and E.In addition, Kenjis etc. find that matter suppresses the rat uveitis (Elu) of endotaxin induction, and its anti-inflammatory action is suitable with Ni Songlong.Particularly Recent study finds, fucoxanthine can burn fat cell significantly, the effect of elimination fat accumulation, thus play significant fat-reducing effect, more make fucoxanthine occupy consequence in diet pill market, therefore, fucoxanthine is a broad-spectrum marine active substance.
At present, more existing research about fucoxanthine extracting method report: as Chinese patent CN1706836A discloses a kind of method being separated fucoxanthine from marine alga, the edible silica gel column chromatography repeatedly of the method is separated, and use dimethyl alum, do not remove by low-temperature reduced-pressure distillating method, sample is difficult to dry under cryogenic, is difficult to obtain high-purity fucoxanthine.Chinese patent CN100999508A discloses a kind of method extracting fucoxanthine from brown alga, and only use macroporous absorption column chromatography for separation, the samples contg of acquisition is 1 ~ 20%, and purity is lower.Chinese patent CN102336725 discloses a kind of method extracting the enriched material containing fucoxanthine from marine alga, the method uses ferment treatment, and extract enriched material and be separated, purity is lower.Chinese patent CN102007216 discloses the preparation method of fucoxanthine and the micro-algae for fucoxanthine, the method is extracted and is prepared fucoxanthine from the micro-algae cultivated, the method relates generally to the cultivation of producing the micro-algae of fucoxanthine, only obtains extracting substance, does not carry out separation and purification.In sum, above preparation method all fails to obtain highly purified fucoxanthine, high-valued Application and Development can not be carried out to fucoxanthine and fucoxanthine series product, therefore, be necessary invention a kind of from brown alga or in other algae extraction and isolation obtain high-purity fucoxanthine, to carry out the high-valued Application and Development of fucoxanthine.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of fucoxanthine, the method extraction and isolation from marine alga obtains.
The present invention includes following steps:
1) by the brown alga material dry after cleaning, with organic solvent dipping, extract;
2) extracting solution that step 1) obtains is concentrated, obtain fucoxanthine crude extract;
3) by step 2) the fucoxanthine crude extract that obtains adopts column chromatography for separation, collects fucoxanthine cut, concentrated, obtains fucoxanthine raw material;
4) by the fucoxanthine raw material organic solvent dissolution of step 3) acquisition, proceed in the sample injection bottle of half preparation/preparative high performance liquid chromatography, startup half preparation/preparative high performance liquid chromatography separation and purification, is detected by on-line ultraviolet or fucoxanthine refined solution collected automatically by mass signal triggering run tank;
5) by the fucoxanthine refined solution concentrating under reduced pressure that step 4) is collected, lyophilize, obtains high-purity fucoxanthine.
In step 1), described brown alga can be selected from the one in sea-tangle, wakame, sargassum thunbergii etc., and described brown alga can adopt the tankage in fresh brown alga and the brown alga course of processing; Described organic solvent can be selected from the one in methyl alcohol, ethanol, acetone, ethyl acetate etc.; Described extraction can adopt organic solvent to carry out flooding or supersound extraction, and extraction time can be 0.1 ~ 48h;
In step 2) in, described concentrating can adopt concentrating under reduced pressure or membrane concentration.
In step 3), be separated silica gel column chromatography can be adopted to carry out initial gross separation, silica gel can be 100 ~ 400 orders, eluent can be methylene chloride-methanol (50 ~ 5: 1), sherwood oil-acetone (20 ~ 3: 1), petroleum ether-ethyl acetate (20 ~ 3: 1), collect fucoxanthine cut, macroporous absorption column chromatography, eluent can be alcohol-water (1: 99 ~ 99: 1), collects alcohol-water (70: 30 ~ 99: 1) elution fraction.
In step 4), described organic solvent can be selected from the one in methyl alcohol, ethanol, acetone, ethyl acetate etc.; The column packing of described half preparation/preparative high performance liquid chromatography can be C8 post or C18 post, and the diameter of half preparation/preparative column can be 5 ~ 50mm; Described ultraviolet on-line checkingi can adopt UV-detector or diode-array detector, and determined wavelength is 400 ~ 500nm, and it take mass spectrum as detector that described mass signal triggers cut, and ion source adopts ESI
+or APCI
+; The sample size of preparation is 50 ~ 5000 μ L/ time; The concentration of volume percent of mobile phase methanol is 70% ~ 95%, and the volume percent of ethanol is 60% ~ 95%, and the volume percent of acetonitrile is 75% ~ 95%; Flow rate of mobile phase is 2 ~ 100mL/min;
In step 5), the temperature of described concentrating under reduced pressure can be 20 ~ 50 DEG C.
Compared with existing method, outstanding advantages of the present invention and technique effect as follows:
After tested, the purity of the fucoxanthine obtained by the present invention is greater than 99%.
Accompanying drawing explanation
Fig. 1 is high performance liquid phase ultraviolet detection color atlas.
Fig. 2 is that high performance liquid phase simpleness detects color atlas.
Fig. 3 is fucoxanthine purity detecting color atlas.
Fig. 4 is fucoxanthine hydrogen nuclear magnetic resonance spectrogram.
Fig. 5 is fucoxanthine carbon-13 nmr spectra spectrum.
Fig. 6 is fucoxanthine ultraviolet spectrogram.
Fig. 7 is fucoxanthine infrared spectrogram.In the figure 7, X-coordinate is wave number Wavenumber (cm
-1), ordinate zou is transmittance Transmittance (%).
Fig. 8 is fucoxanthine mass spectrum.
Fig. 9 is fucoxanthine high resolution mass spectrum figure.
Embodiment
Following examples will the present invention is further illustrated by reference to the accompanying drawings.
Embodiment 1
(1) extraction of raw material: get sea-tangle 1kg, cleans, puts shady and cool place and dry, add methyl alcohol 5L, supersound extraction 30min, filters, extracts 2 times, united extraction liquid, concentrating under reduced pressure with method, obtains fucoxanthine medicinal extract;
(2) initial gross separation purifying: adopt macroporous adsorbent resin to carry out column chromatography, carry out wash-out with the aqueous solution, 30% aqueous ethanolic solution, 60% aqueous ethanolic solution, 90% aqueous ethanolic solution respectively, collect 90% aqueous ethanolic solution wash-out part, concentrate drying, obtain fucoxanthine crude product 260mg, content 25%;
(3) fucoxanthine is refined:
A) preparation of raw material: solution fucoxanthine crude product being mixed with 5mg/ml;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C8 post (250mm × 10mm), and flow phase system is 70% methanol aqueous solution, and flow rate of mobile phase is 10mL/min, and determined wavelength is 450nm;
D) sampling volume: 300 μ L;
Preparing liquid 350mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthine, concentrated solution being carried out lyophilize, obtains fucoxanthine 53mg, purity 98.99%.
Embodiment 2
1) extraction of raw material: get sea-tangle 1kg, cleans, puts shady and cool place and dry, add ethanol 5L, supersound extraction 60min, filters, extracts 2 times, united extraction liquid, concentrating under reduced pressure with method, obtains fucoxanthine medicinal extract;
2) initial gross separation purifying: adopt macroporous adsorbent resin to carry out column chromatography, wash-out is carried out respectively with the aqueous solution, 30% aqueous ethanolic solution, 60% aqueous ethanolic solution, 80% aqueous ethanolic solution, 100% aqueous ethanolic solution, collect 100% aqueous ethanolic solution wash-out part, concentrate drying, obtain fucoxanthine crude product 150mg, content 53%;
3) fucoxanthine is refined:
A) preparation of raw material: solution fucoxanthine crude product being mixed with 5mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C8 post (250mm × 10mm), and flow phase system is 85% methanol aqueous solution, and flow rate of mobile phase is 10mL/min, and determined wavelength is 450nm;
D) sampling volume: 300 μ L;
Preparing liquid 310mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthine, concentrated solution being carried out lyophilize, obtains fucoxanthine 65mg, purity 99.09%.
Embodiment 3
1) extraction of raw material: get sea-tangle 1kg, cleans, puts shady and cool place and dry, add acetone 5L, supersound extraction 30min, filters, extracts 2 times, united extraction liquid, concentrating under reduced pressure with method, obtains fucoxanthine medicinal extract;
2) initial gross separation purifying: adopt silica gel column chromatography, use methylene chloride-methanol (50: 1,25: 1,10: 1,5: 1) to carry out wash-out respectively, collects (25: 1 and 10: 1) elution fraction, concentrate drying, obtains fucoxanthine crude product 105mg, content 87%;
3) fucoxanthine is refined:
A) preparation of raw material: solution fucoxanthine crude product being mixed with 5mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C8 post (250mm × 10mm), and flow phase system is 95% methanol aqueous solution, and flow rate of mobile phase is 10mL/min, and determined wavelength is 450nm;
D) sampling volume: 400 μ L;
Preparing liquid 190mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthine, concentrated solution being carried out lyophilize, obtains fucoxanthine 69mg, purity 99.39%.
Embodiment 4
1) extraction of raw material: get sea-tangle 1kg, cleans, puts shady and cool place and dry, add ethyl acetate 5L, supersound extraction 30min, filters, extracts 2 times, united extraction liquid, concentrating under reduced pressure with method, obtains fucoxanthine medicinal extract;
2) initial gross separation purifying: adopt silica gel column chromatography, use sherwood oil-acetone (20: 1,10: 1,5: 1,3: 1) to carry out wash-out respectively, collects (10: 1 and 5: 1) elution fraction, concentrate drying, obtains fucoxanthine crude product 115mg, content 83%;
3) fucoxanthine is refined:
A) preparation of raw material: solution fucoxanthine crude product being mixed with 10mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C8 post (250mm × 10mm), and flow phase system is 90% aqueous ethanolic solution, and flow rate of mobile phase is 10mL/min, and determined wavelength is 450nm;
D) sampling volume: 400 μ L;
Preparing liquid 130mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthine, concentrated solution being carried out lyophilize, obtains fucoxanthine 62mg, purity 99.25%.
Embodiment 5
(1) extraction of raw material: get sea-tangle 1kg, clean, put shady and cool place and dry, add methylene chloride 5L, supersound extraction 30min, filters, extract 2 times, united extraction liquid, concentrating under reduced pressure with method, obtains fucoxanthine medicinal extract;
(2) initial gross separation purifying: adopt silica gel column chromatography, use petroleum ether-ethyl acetate (20: 1,10: 1,5: 1,3: 1) to carry out wash-out respectively, collect (10: 1 and 5: 1) elution fraction, concentrate drying, obtain fucoxanthine crude product 105mg, content 89%;
(3) fucoxanthine is refined:
A) preparation of raw material: solution fucoxanthine crude product being mixed with 10mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C8 post (250mm × 10mm), and flow phase system is 85% acetonitrile solution, and flow rate of mobile phase is 10mL/min, and determined wavelength is 450nm;
D) sampling volume: 400 μ L;
Preparing liquid 120mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthine, concentrated solution being carried out lyophilize, obtains fucoxanthine 62mg, purity 99.28%.
Embodiment 6
1) extraction of raw material: get wakame 5kg, cleans, puts shady and cool place and dry, add methyl alcohol 25L, supersound extraction 30min, filters, extracts 2 times, united extraction liquid, concentrating under reduced pressure with method, obtains fucoxanthine medicinal extract;
2) initial gross separation purifying: adopt macroporous adsorbent resin to carry out column chromatography, carry out wash-out with the aqueous solution, 30% aqueous ethanolic solution, 60% aqueous ethanolic solution, 90% aqueous ethanolic solution respectively, collect 90% aqueous ethanolic solution wash-out part, concentrate drying, obtain fucoxanthine crude product 1000mg, content 38%;
3) fucoxanthine is refined:
A) preparation of raw material: solution fucoxanthine crude product being mixed with 10mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C8 post (250mm × 20mm), and flow phase system is 80% methanol aqueous solution, and flow rate of mobile phase is 20mL/min, and determined wavelength is 450nm;
D) sampling volume: 700 μ L;
Preparing liquid 1050mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthine, concentrated solution being carried out lyophilize, obtains fucoxanthine 278mg, purity 99.06%.
Embodiment 7
1) extraction of raw material: get wakame 5kg, cleans, puts shady and cool place and dry, add ethanol 25L, supersound extraction 60min, filters, extracts 2 times, united extraction liquid, concentrating under reduced pressure with method, obtains fucoxanthine medicinal extract;
2) initial gross separation purifying: adopt macroporous adsorbent resin to carry out column chromatography, wash-out is carried out respectively with the aqueous solution, 30% aqueous ethanolic solution, 60% aqueous ethanolic solution, 80% aqueous ethanolic solution, 100% aqueous ethanolic solution, collect 100% aqueous ethanolic solution wash-out part, concentrate drying, obtain fucoxanthine crude product 630mg, content 67%;
3) fucoxanthine is refined:
A) preparation of raw material: solution fucoxanthine crude product being mixed with 15mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C8 post (250mm × 20mm), and flow phase system is 85% methanol aqueous solution, and flow rate of mobile phase is 20mL/min, and determined wavelength is 450nm;
D) sampling volume: 800 μ L;
Preparing liquid 890mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthine, concentrated solution being carried out lyophilize, obtains fucoxanthine 299mg, purity 99.17%.
Embodiment 8
1) extraction of raw material: get wakame 10kg, cleans, puts shady and cool place and dry, add acetone 50L, supersound extraction 30min, filters, extracts 2 times, united extraction liquid, concentrating under reduced pressure with method, obtains fucoxanthine medicinal extract;
2) initial gross separation purifying: adopt silica gel column chromatography, use methylene chloride-methanol (50: 1,25: 1,10: 1,5: 1) to carry out wash-out respectively, collects (25: 1 and 10: 1) elution fraction, concentrate drying, obtains fucoxanthine crude product 1150mg, content 89%;
3 fucoxanthines are refined:
A) preparation of raw material: solution fucoxanthine crude product being mixed with 15mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C8 post (250mm × 20mm), and flow phase system is 95% methanol aqueous solution, and flow rate of mobile phase is 25mL/min, and determined wavelength is 450nm;
D) sampling volume: 800 μ L;
Preparing liquid 1560mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthine, concentrated solution being carried out lyophilize, obtains fucoxanthine 787mg, purity 99.38%.
Embodiment 9
1) extraction of raw material: get wakame 20kg, cleans, puts shady and cool place and dry, add ethyl acetate 80L, and dipping 24h, filters, extract 2 times, united extraction liquid, concentrating under reduced pressure with method, obtains fucoxanthine medicinal extract;
2) initial gross separation purifying: adopt silica gel column chromatography, use sherwood oil-acetone (20: 1,10: 1,5: 1,3: 1) to carry out wash-out respectively, collects (10: 1 and 5: 1) elution fraction, concentrate drying, obtains fucoxanthine crude product 1889mg, content 87%;
3) fucoxanthine is refined:
A) preparation of raw material: solution fucoxanthine crude product being mixed with 20mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C18 post (250mm × 20mm), and flow phase system is 90% aqueous ethanolic solution, and flow rate of mobile phase is 10mL/min, and determined wavelength is 450nm;
D) sampling volume: 1000 μ L;
Preparing liquid 2150mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthine, concentrated solution being carried out lyophilize, obtains fucoxanthine 1376mg, purity 99.48%.
Embodiment 10
1) extraction of raw material: get sargassum thunbergii 20kg, clean, put shady and cool place and dry, add methylene chloride 80L, Soakage extraction 24h, filters, extract 2 times, united extraction liquid, concentrating under reduced pressure with method, obtains fucoxanthine medicinal extract;
2) initial gross separation purifying: adopt silica gel column chromatography, use petroleum ether-ethyl acetate (20: 1,10: 1,5: 1,3: 1) to carry out wash-out respectively, collects (10: 1 and 5: 1) elution fraction, concentrate drying, obtains fucoxanthine crude product 1965mg, content 85%;
3) fucoxanthine is refined:
A) preparation of raw material: solution fucoxanthine crude product being mixed with 20mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C18 post (250mm × 20mm), and flow phase system is 85% acetonitrile solution, and flow rate of mobile phase is 10mL/min, and determined wavelength is 450nm;
D) sampling volume: 3000 μ L;
Preparing liquid 1580mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthine, concentrated solution being carried out lyophilize, obtains fucoxanthine 1480mg, purity 99.55%.
Embodiment 11
1) extraction of raw material: get sargassum thunbergii 20kg, cleans, puts shady and cool place and dry, add methyl alcohol 80L, and dipping 24h, filters, extract 2 times, united extraction liquid, concentrating under reduced pressure with method, obtains fucoxanthine medicinal extract;
2) initial gross separation purifying: adopt silica gel column chromatography, use sherwood oil-acetone (20: 1,10: 1,5: 1,3: 1) to carry out wash-out respectively, collects (10: 1 and 5: 1) elution fraction, concentrate drying, obtains fucoxanthine crude product 1850mg, content 85%;
3) fucoxanthine is refined:
A) preparation of raw material: solution fucoxanthine crude product being mixed with 20mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C18 post (250mm × 30mm), and flow phase system is 90% aqueous ethanolic solution, and flow rate of mobile phase is 10mL/min, and determined wavelength is 450nm;
D) sampling volume: 4000 μ L;
Preparing liquid 2150mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthine, concentrated solution being carried out lyophilize, obtains fucoxanthine 1476mg, purity 99.68%.
Embodiment 12
1) extraction of raw material: get sargassum thunbergii 20kg, cleans, puts shady and cool place and dry, add ethanol 80L, Soakage extraction 24h, filters, extracts 2 times, united extraction liquid, concentrating under reduced pressure with method, obtains fucoxanthine medicinal extract;
2) initial gross separation purifying: adopt silica gel column chromatography, use petroleum ether-ethyl acetate (20: 1,10: 1,5: 1,3: 1) to carry out wash-out respectively, collects (10: 1 and 5: 1) elution fraction, concentrate drying, obtains fucoxanthine crude product 1795mg, content 87%;
3) fucoxanthine is refined:
A) preparation of raw material: solution fucoxanthine crude product being mixed with 20mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C18 post (250mm × 30mm), and flow phase system is 85% acetonitrile solution, and flow rate of mobile phase is 10mL/min, and determined wavelength is 450nm;
D) sampling volume: 5000 μ L;
Preparing liquid 1080mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthine, concentrated solution being carried out lyophilize, obtains fucoxanthine 1493mg, purity 99.59%.
Embodiment 13
1) extraction of raw material: get sargassum thunbergii 1kg, cleans, puts shady and cool place and dry, add methyl alcohol 5L, supersound extraction 30min, filters, extracts 2 times, united extraction liquid, concentrating under reduced pressure with method, obtains fucoxanthine medicinal extract;
2) initial gross separation purifying: adopt macroporous adsorbent resin to carry out column chromatography, carry out wash-out with the aqueous solution, 30% aqueous ethanolic solution, 60% aqueous ethanolic solution, 90% aqueous ethanolic solution respectively, collect 90% aqueous ethanolic solution wash-out part, concentrate drying, obtain fucoxanthine crude product 260mg, content 25%;
3) fucoxanthine is refined:
A) preparation of raw material: solution fucoxanthine crude product being mixed with 5mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C8 post (250mm × 10mm), and flow phase system is 70% methanol aqueous solution, and flow rate of mobile phase is 10mL/min; Mass Spectrometry Conditions: ESI
+corona current 3.0 μ A, one-level taper hole voltage 30V, secondary taper hole voltage 3.0V, source temperature 120 DEG C, desolventizing temperature 350 DEG C, desolventizing nitrogen flow rate 300L/h, taper hole nitrogen flow rate 50L/h, height total mass number resolving power is respectively 15, ion energy 0.5, mass scanning pattern is selected in the data gathering of matter scanning spectrum, and sweep limit is 500 ~ 800;
D) sampling volume: 300 μ L;
Preparing liquid 350mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthine, concentrated solution being carried out lyophilize, obtains fucoxanthine 53mg, purity 99.02%.
Embodiment 14
1) extraction of raw material: get siliquosa Pelvetia 1kg, cleans, puts shady and cool place and dry, add ethanol 5L, supersound extraction 60min, filters, extracts 2 times, united extraction liquid, concentrating under reduced pressure with method, obtains fucoxanthine medicinal extract;
2) initial gross separation purifying: adopt macroporous adsorbent resin to carry out column chromatography, wash-out is carried out respectively with the aqueous solution, 30% aqueous ethanolic solution, 60% aqueous ethanolic solution, 80% aqueous ethanolic solution, 100% aqueous ethanolic solution, collect 100% aqueous ethanolic solution wash-out part, concentrate drying, obtain fucoxanthine crude product 150mg, content 53%;
3) fucoxanthine is refined:
A) preparation of raw material: solution fucoxanthine crude product being mixed with 5mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C8 post (250mm × 10mm), and flow phase system is 85% methanol aqueous solution, and flow rate of mobile phase is 10mL/min; Mass Spectrometry Conditions: ESI
+corona current 3.0 μ A, one-level taper hole voltage 30V, secondary taper hole voltage 3.0V, source temperature 120 DEG C, desolventizing temperature 350 DEG C, desolventizing nitrogen flow rate 300L/h, taper hole nitrogen flow rate 50L/h, height total mass number resolving power is respectively 15, ion energy 0.5, mass scanning pattern is selected in the data gathering of matter scanning spectrum, and sweep limit is 500 ~ 800;
D) sampling volume: 300 μ L;
Preparing liquid 310mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthine, concentrated solution being carried out lyophilize, obtains fucoxanthine 65mg, purity 99.09%.
Embodiment 15
1) extraction of raw material: get siliquosa Pelvetia 1kg, cleans, puts shady and cool place and dry, add acetone 5L, supersound extraction 30min, filters, extracts 2 times, united extraction liquid, concentrating under reduced pressure with method, obtains fucoxanthine medicinal extract;
2) initial gross separation purifying: adopt silica gel column chromatography, use methylene chloride-methanol (50:1,25:1,10:1:5:1) to carry out wash-out respectively, collects (25:1 and 10:1) elution fraction, concentrate drying, obtains fucoxanthine crude product 105mg, content 87%;
3) fucoxanthine is refined:
A) preparation of raw material: solution fucoxanthine crude product being mixed with 5mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C8 post (250mm × 10mm), flow phase system is 95% methanol aqueous solution, flow rate of mobile phase is 10mL/min, Mass Spectrometry Conditions: APCI+, corona current 3.0 μ A, one-level taper hole voltage 30V, secondary taper hole voltage 3.0V, source temperature 120 DEG C, desolventizing temperature 350 DEG C, desolventizing nitrogen flow rate 300L/h, taper hole nitrogen flow rate 50L/h, height total mass number resolving power is respectively 15, ion energy 0.5, mass scanning pattern is selected in the data gathering of matter scanning spectrum, and sweep limit is 500 ~ 800;
D) sampling volume: 400 μ L;
Preparing liquid 190mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthine, concentrated solution being carried out lyophilize, obtains fucoxanthine 69mg, purity 99.39%.
The structural formula of the fucoxanthine of gained is as follows:
High performance liquid phase ultraviolet detection color atlas is see Fig. 1, high performance liquid phase simpleness detects color atlas see Fig. 2, fucoxanthine purity detecting color atlas is see Fig. 3, fucoxanthine hydrogen nuclear magnetic resonance spectrogram is see Fig. 4, fucoxanthine carbon-13 nmr spectra spectrogram is see Fig. 5, and fucoxanthine ultraviolet spectrogram is see Fig. 6, and fucoxanthine infrared spectrogram is see Fig. 7, fucoxanthine mass spectrum is see Fig. 8, and fucoxanthine high resolution mass spectrum figure is see Fig. 9.
Claims (5)
1. the preparation method of fucoxanthine, is characterized in that comprising the following steps:
1) by the brown alga material dry after cleaning, with organic solvent dipping, extract, extraction time is 0.1 ~ 48h;
2) by step 1) extracting solution that obtains concentrates, and obtains fucoxanthine crude extract;
3) by step 2) the fucoxanthine crude extract that obtains adopts column chromatography for separation, collects fucoxanthine cut, concentrated, obtains fucoxanthine raw material; Described column chromatography for separation adopts silica gel column chromatography to carry out initial gross separation, silica gel is 100 ~ 400 orders, and eluent is sherwood oil-acetone or petroleum ether-ethyl acetate, and the proportioning of sherwood oil and acetone is 20 ~ 3: 1, the proportioning of sherwood oil and ethyl acetate is 20 ~ 3: 1, collects fucoxanthine cut; Or described column chromatography for separation adopts macroporous adsorbent resin to carry out column chromatography, and eluent is alcohol-water, and collect alcohol-water elution fraction, the proportioning of ethanol and water is 1: 99 ~ 99: 1, and the proportioning of collecting ethanol and water is 70: 30 ~ 99: 1;
4) by step 3) the fucoxanthine raw material organic solvent dissolution that obtains, proceed in the sample injection bottle of half preparation/preparative high performance liquid chromatography, startup half preparation/preparative high performance liquid chromatography separation and purification, is detected by on-line ultraviolet or fucoxanthine refined solution collected automatically by mass signal triggering run tank;
5) by step 4) the fucoxanthine refined solution concentrating under reduced pressure collected, lyophilize, obtains high-purity fucoxanthine;
Step 1) and step 4) in, described organic solvent is selected from the one in methyl alcohol, ethanol, acetone, ethyl acetate; Step 4) described in the column packing of half preparation/preparative high performance liquid chromatography be C8 post or C18 post, partly the diameter of preparation/preparative column is 5 ~ 50mm; Step 4) in, described ultraviolet on-line checkingi adopts UV-detector or diode-array detector, and determined wavelength is 400 ~ 500nm, and it take mass spectrum as detector that described mass signal triggers cut, and ion source adopts ESI
+or APCI
+; The sample size of preparation is 50 ~ 5000 μ L/ time; The concentration of volume percent of mobile phase methanol is 70% ~ 95%, and the volume percent of ethanol is 60% ~ 95%, and the volume percent of acetonitrile is 75% ~ 95%; Flow rate of mobile phase is 2 ~ 100mL/min.
2. the preparation method of fucoxanthine as claimed in claim 1, is characterized in that in step 1) in, described brown alga is selected from the one in sea-tangle, wakame, sargassum thunbergii.
3. the preparation method of fucoxanthine as claimed in claim 1 or 2, is characterized in that described brown alga adopts the tankage in fresh brown alga and the brown alga course of processing.
4. the preparation method of fucoxanthine as claimed in claim 1, is characterized in that in step 2) in, described concentrated employing concentrating under reduced pressure or membrane concentration.
5. the preparation method of fucoxanthine as claimed in claim 1, is characterized in that in step 5) in, the temperature of described concentrating under reduced pressure is 20 ~ 50 DEG C.
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| JP2013166790A (en) * | 2013-05-30 | 2013-08-29 | Shingen Medical Co Ltd | Method for producing fucoxanthin |
| CN104327017B (en) * | 2014-11-25 | 2016-01-20 | 武汉大学 | A kind of Isolation and purification method of brown alga flavine |
| JP6399920B2 (en) * | 2014-12-19 | 2018-10-03 | ライオン株式会社 | Body fat reducing agent, weight loss promoting agent, blood neutral fat reducing agent and blood free fatty acid reducing agent |
| CN106083765B (en) * | 2016-05-26 | 2018-07-03 | 深圳市海立方生物科技有限公司 | A kind of Environmental Safety cleaning agent with antibacterial functions |
| CN106478554A (en) * | 2016-10-17 | 2017-03-08 | 青岛博恩高科生物技术有限公司 | A kind of method extracting fucoxanthine and Sargassum polysaccharides comprehensive from Sargassum |
| CN106727729A (en) * | 2016-12-18 | 2017-05-31 | 钦州学院 | A kind of method that fucoxanthine and fucoidin crude product are comprehensively extracted from marine alga |
| CN108530397B (en) * | 2018-05-07 | 2022-07-12 | 中国科学院海洋研究所 | Preparation of sargassum horneri algae powder and method for extracting fucoxanthin in sargassum horneri algae powder |
| CN111153874B (en) * | 2020-02-19 | 2022-11-18 | 自然资源部第一海洋研究所 | Method for extracting fucoxanthin from seaweed by utilizing four-region simulated moving bed system |
| CN115109013A (en) * | 2021-03-19 | 2022-09-27 | 宁波坤行健医药科技有限公司 | A method for extracting fucoidan and fucoxanthin from brown algae |
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