CN104789611A - Preparation method of fucoxanthol - Google Patents
Preparation method of fucoxanthol Download PDFInfo
- Publication number
- CN104789611A CN104789611A CN201510147401.4A CN201510147401A CN104789611A CN 104789611 A CN104789611 A CN 104789611A CN 201510147401 A CN201510147401 A CN 201510147401A CN 104789611 A CN104789611 A CN 104789611A
- Authority
- CN
- China
- Prior art keywords
- fucoxanthol
- preparation
- solution
- obtains
- purity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Epoxy Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a preparation method of fucoxanthol. The preparation method comprises the following steps: under the situation of adding a catalyst, enzymolyzing fucoxanthin by adopting lipase, reacting, generating fucoxanthol, concentrating and drying, extracting through methanol and concentrating to obtain a crude fucoxanthol extract; enabling the crude fucoxanthol extract to be used as a solution medium in a form of a mobile phase, configuring into preparation liquid to be separated, transferring the preparation liquid to be separated into a semi-preparative/preparative efficient liquid phase chromatography sampling bottle and starting a semi-preparative/preparative efficient liquid phase chromatography, wherein the mobile phase is selected from one of a methanol-water solution, an ethanol-water solution or an acetonitrile-water solution; balancing a chromatographic column, automatically sampling, separating and purifying the fucoxanthol, triggering a fraction collector through ultraviolet on-line detection and in combination with mass spectrometry detection signals, automatically collecting fucoxanthol purification liquid, and decompressing, condensing, freezing and drying the purification liquid to obtain the fucoxanthol of which the purity is larger than 99 percent. The preparation method is simple in operation, high in yield, suitable for automatic control and preparation of the high-purity fucoxanthol.
Description
Technical field
The present invention relates to a kind of preparation method of high-purity fucoxanthol, belong to foods processing technique and functional health field.
Background technology
Fucoxanthine (fucoxanthin) is also known as brown alga flavine, in edible brown alga, as wakame (Alariaceae, Undaria pinnatifida), the natural carotenoid extracted in sea-tangle (Laminaria japonica Aresch), has 5, the 6-epoxy unsaturation propadiene bond structures that a chemical property is active respectively at the two ends of its rigidity alltrans long-chain, because of and differ from other carotenoid molecules, there is very strong biological activity.Its various biological activity is proved in recent years, 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.
Fucoxanthol is the similar of the meta-bolites that fucoxanthine sloughs an ethanoyl, its structure and fucoxanthine, has very strong biological activity, comprises the multiple biological activitys such as antitumor, anti-inflammatory, anti-oxidant, fat-reducing.Recent study shows, can be translated into fucoxanthol and be utilized after Mouse oral fucoxanthine.The research such as Hayato Maeda also finds that the fucoxanthine meta-bolites fucoxanthol in 3T3-L1 clone by reducing PPAR γ (Peroxisome Proliferator-activated Receptors) thus reaching the object suppressing 3T3-L1 preadipocyte to be tied to adipose cell lines differentiation, thus can reach the effect of fat-reducing.There are some researches show, the anti tumor activity in vitro of fucoxanthol is even better than fucoxanthine.Domestic only a few producer extracts and prepares low levels fucoxanthine raw material (HPLC method at present, content < 1%), outlet is American-European, Japan and other countries, there is not yet the report of fucoxanthol batch preparation research at present both at home and abroad, high-valued Application and Development can not be carried out to fucoxanthol and fucoxanthol series product, therefore, the preparation method inventing a kind of high-purity fucoxanthol is necessary.
Summary of the invention
The object of the present invention is to provide a kind of is substrate with fucoxanthine, prepares the method that fucoxanthol obtains fucoxanthol crude extract, also provide a kind of and fucoxanthol crude extract is carried out the method that separation and purification obtains high-purity sample again by enzyme digestion reaction.
One aspect of the present invention relates to a kind of preparation method of fucoxanthine, it is characterized in that, comprises the following steps:
(1), with fucoxanthine high-purity monomer or with fucoxanthine extract for raw material;
(2), by raw material and lipase, catalyst mix, react in water-bath with after PBS buffer solution;
Preferably; described catalyzer is lipase accelerator, the Sodium cholic acid salts substances such as preferred sodium deoxycholate, Taurocholic acid sodium salt, Sodium cholic acid, NaGC, deoxidation Taurocholic acid sodium salt, Taurocholic acid sodium salt salt hydrate, sweet ammonia SODIUM CHENODIOL, gallodesoxycholic acid sodium, sodium taurochenodeoxycholate.
Preferably, described lipase is: the lipase such as pig pancreatic lipases, ox pancreas lipase, lipase from candida sp.
Preferably, step 2) described in by raw material and lipase, catalyst mix, raw material: lipase: the ratio of catalyzer is 1:1:1 to 1:40:40,1:1:1 can be selected from, 1:3:2,1:5:5,1:4:2,2:5:3,5:4:4,1:2:2,1:3:3,2:10:5,1:10:10,1:20:20,1:40:40;
Preferably, step 2) described in the pH value of PBS damping fluid be 3-11, the temperature of water-bath is 20-70 DEG C, and the reaction times is 0-24h; PH value is selected from 4,5,6,7,8,9,10,11; Bath temperature is selected from 20 DEG C, 30 DEG C, 37 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C; Reaction times is selected from 0.5h, 1h, 2h, 4h, 8h, 10h, 12h, 16h, 20h, 24h.
Preferably, in step 1) in adopt raw material be high-purity fucoxanthine monomer or fucoxanthine extract,
Purity is 1-99%.
Preferably, the preparation method of described fucoxanthine, also comprises 3), by step 2) reaction solution that obtains carries out being concentrated into dry, use methyl alcohol supersound extraction, extracting solution is concentrated into dry, obtains fucoxanthol crude extract.
The preparation method of fucoxanthol of the present invention, selectablely comprises step 4), by step 3) crude extract that obtains adopts column chromatography to carry out initial gross separation, collects fucoxanthol cut, concentratedly obtains fucoxanthol coarse raw materials.When step 1) in raw material used when being fucoxanthine extract, preferably include this step.
Preferably, step 4) described in column chromatography be silica gel column chromatography or macroporous adsorbent resin column chromatography.
Preferably, step 4) described in silica gel column chromatography, silica gel granularity is 100-400 order, and eluent is methylene chloride-methanol (50:1-5:1), or sherwood oil-acetone (20:1-3:1), or petroleum ether-ethyl acetate (20:1-3:1).Preferably use methylene chloride-methanol (50:1,25:1,10:1:5:1) to carry out wash-out respectively, collect (25:1 and 10:1) elution fraction; Preferably use sherwood oil-acetone (20:1-3:1) to carry out wash-out respectively, collect (25:1 and 10:1) elution fraction; Preferably use petroleum ether-ethyl acetate (20:1-3:1) to carry out wash-out respectively, collect (25:1 and 10:1) elution fraction.
Preferably, step 4) described in macroporous absorption column chromatography, filler used is that AB-8, D101 etc. are wherein a kind of, and eluent is alcohol-water (1:99-99:1).
Preferably, the preparation method of described fucoxanthol, also comprise step 5), by step 3) the fucoxanthol crude extract that obtains or step 4) the fucoxanthol coarse raw materials moving phase that obtains dissolves, proceed in half preparation/preparative high performance liquid chromatography sample injection bottle, start half preparation/preparative high performance liquid chromatography and carry out separation and purification, trigger run tank by on-line ultraviolet detection signal and automatically collect fucoxanthol refined solution.
Preferably, step 5) described in the moving phase of half preparation/preparative high performance liquid chromatography employing be respectively one in methanol aqueous solution, aqueous ethanolic solution or acetonitrile solution, the concentration of volume percent of methanol aqueous solution is 70-95%, the concentration of volume percent of aqueous ethanolic solution is 60-95%, and the concentration of volume percent of acetonitrile solution is 55-95%; The concentration of volume percent of methanol aqueous solution is selected from: 70%, 80%, 90%, 95%; The concentration of volume percent of aqueous ethanolic solution is selected from: 60%, 70%, 80%, 90%, 95%; The concentration of volume percent of acetonitrile solution is 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%.
Preferably, step 5) the partly preparation/preparative column filler that adopts of described half preparation/preparative high performance liquid chromatography is C8 post or C18 post.
Preferably, step 5) diameter of partly preparation/preparative column that adopts of described half preparation/preparative high performance liquid chromatography is 10-50mm.
Preferably, step 5) described in the sample size that adopts of half preparation/preparative high performance liquid chromatography be that 100 μ L/-20mL/ is secondary.
Preferably, step 5) described in the flow rate of mobile phase of half preparation/preparative high performance liquid chromatography employing be 5-200mL/min, preferably 5,6,10,20,28,50,80,100,120,150,180,200mL/min.
Preferably, step 5) described in the detector of half preparation/preparative high performance liquid chromatography employing be UV-detector or diode-array detector, determined wavelength is 400-500nm, preferred 450nm.
Preferably, the preparation method of described fucoxanthol, also comprises step 6), by step 5) in fucoxanthol refined solution carry out concentrating under reduced pressure after, get concentrated solution and carry out lyophilize, obtain high-purity fucoxanthol.
Preferably, step 6) described concentrating under reduced pressure temperature is 20-50 DEG C, drying mode is lyophilize.
Preferably, step 6) described high-purity fucoxanthol, purity is greater than 99%.
Accompanying drawing explanation
Fucoxanthol preparative chromatography figure in Fig. 1 embodiment 1.
Fucoxanthol purity detecting color atlas in Fig. 2 embodiment 1.
The hydrogen nuclear magnetic resonance spectrogram of the fucoxanthol prepared by Fig. 3 embodiment 1.
The carbon-13 nmr spectra figure of the fucoxanthol prepared by Fig. 4 embodiment 1.
Embodiment
Embodiment 1
1) enzyme digestion reaction: get fucoxanthine (purity is 99%) 50mg, add lipase 50mg, Taurocholic acid sodium salt 50mg, is dissolved in the 0.1M PBS buffered soln of pH=3, react 1h at 20 DEG C after, be concentrated into dry, add the methyl alcohol supersound extraction of 50mL, filter, filtrate is concentrated into dry, obtain fucoxanthol reactant 55mg, the transformation efficiency of fucoxanthol is 23%;
2) fucoxanthol is refined:
A) configuration of raw material: solution fucoxanthine crude product being configured to 6mg/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, flow rate of mobile phase is 5mL/min, and determined wavelength is 450nm;
D) sampling volume: 100 μ L;
Preparing liquid 100mL, concentrating under reduced pressure at 30 DEG C by running Semipreparative chromatography acquisition fucoxanthol, concentrated solution being carried out lyophilize, obtains fucoxanthol 8mg, purity 99.03%.Embodiment 2
1) enzyme digestion reaction: get fucoxanthine (purity is 99%) 50mg, add lipase 150mg, Taurocholic acid sodium salt 100mg, is dissolved in the 0.1M PBS buffered soln of pH=7, react 1h at 37 DEG C after, be concentrated into dry, add the methyl alcohol supersound extraction of 50mL, filter, filtrate is concentrated into dry, obtain fucoxanthol reactant 85mg, fucoxanthol transformation efficiency 88%;
2) fucoxanthol is refined:
A) configuration of raw material: solution fucoxanthol crude product being configured to 6mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C8 post (250mm × 10mm), and flow phase system is 80% methanol aqueous solution, and flow rate of mobile phase is 6mL/min, and determined wavelength is 450nm;
D) sampling volume: 300 μ L;
Preparing liquid 150mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthol, concentrated solution being carried out lyophilize, obtains fucoxanthol 33mg, purity 99.36%.
Embodiment 3
1) enzyme digestion reaction: get fucoxanthine (purity is 99%) 50mg, add lipase 500mg, Taurocholic acid sodium salt 500mg, is dissolved in the 0.1M PBS buffered soln of pH=11, react 0.5h at 70 DEG C after, be concentrated into dry, add the methyl alcohol supersound extraction of 50mL, filter, filtrate is concentrated into dry, obtain fucoxanthol reactant 88mg, fucoxanthol transformation efficiency 55%;
2) fucoxanthol is refined:
A) configuration of raw material: solution fucoxanthol crude product being configured to 6mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C18 post (250mm × 10mm), and flow phase system is that 90% methyl alcohol is water-soluble, and flow rate of mobile phase is 6mL/min, and determined wavelength is 450nm;
D) sampling volume: 300 μ L;
Preparing liquid 70mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthol, concentrated solution being carried out lyophilize, obtains fucoxanthol 18mg, purity 99.05%.
Embodiment 4
1) enzyme digestion reaction: get fucoxanthine (purity is 99%) 50mg, add lipase 200mg, Taurocholic acid sodium salt 100mg, is dissolved in the 0.1M PBS buffered soln of pH=7, react 24h at 20 DEG C after, be concentrated into dry, add the methyl alcohol supersound extraction of 50mL, filter, filtrate is concentrated into dry, obtain fucoxanthol reactant 83mg, fucoxanthol transformation efficiency 59%;
2) fucoxanthol is refined:
A) configuration of raw material: solution fucoxanthol crude product being configured to 6mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C18 post (250mm × 10mm), and flow phase system is 95% methanol aqueous solution, and flow rate of mobile phase is 6mL/min, and determined wavelength is 450nm;
D) sampling volume: 300 μ L;
Preparing liquid 65mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthol, concentrated solution being carried out freezing, dry, obtaining fucoxanthol 28mg, purity 99.15%.Embodiment 5
1) enzyme digestion reaction: get fucoxanthine (purity is 99%) 50mg, add lipase 200mg, Taurocholic acid sodium salt 100mg, is dissolved in the 0.05M PBS buffered soln of pH=7, react 24h at 20 DEG C after, be concentrated into dry, add the methyl alcohol supersound extraction of 50mL, filter, filtrate is concentrated into dry, obtain fucoxanthol reactant 82mg, fucoxanthol transformation efficiency 53%;
2) fucoxanthol is refined:
A) configuration of raw material: solution fucoxanthol crude product being configured to 6mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C18 post (250mm × 10mm), and flow phase system is 95% methanol aqueous solution, and flow rate of mobile phase is 6mL/min, and determined wavelength is 450nm;
D) sampling volume: 300 μ L;
Preparing liquid 80mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthol, concentrated solution being carried out lyophilize, obtains fucoxanthol 23mg, purity 99.23%.
Embodiment 6
1) enzyme digestion reaction: get fucoxanthine crude product (purity is 50%) 100mg, add lipase 100mg, Taurocholic acid sodium salt 100mg, be dissolved in the 0.05M PBS buffered soln of pH=7, react 2h at 20 DEG C after, be concentrated into dry, add the methyl alcohol supersound extraction of 50mL, filter, filtrate is concentrated into dry, obtains fucoxanthol reactant 167mg;
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, collect (25:1 and 10:1) elution fraction, concentrate drying, obtain fucoxanthol crude product 55mg, content 73%;
3) fucoxanthol is refined:
A) configuration of raw material: solution fucoxanthol crude product being configured to 6mg/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 6mL/min, and determined wavelength is 450nm;
D) sampling volume: 300 μ L;
Preparing liquid 150mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthol, concentrated solution being carried out lyophilize, obtains fucoxanthol 23mg, purity 99.13%.
Embodiment 7
1) enzyme digestion reaction: get fucoxanthine crude product (purity is 25%) 200mg, add lipase 500mg, Taurocholic acid sodium salt 300mg, be dissolved in the 0.05M PBS buffered soln of pH=7, react 2h at 37 DEG C after, be concentrated into dry, add the methyl alcohol supersound extraction of 50mL, filter, filtrate is concentrated into dry, obtains fucoxanthol reactant 367mg;
2) initial gross separation purifying: adopt silica gel column chromatography, use sherwood oil-acetone (20:1-3:1) to carry out wash-out respectively, collects (25:1 and 10:1) elution fraction, concentrate drying, obtains fucoxanthol crude product 79mg, content 47%;
3) fucoxanthol is refined:
A) configuration of raw material: solution fucoxanthol crude product being configured to 6mg/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 6mL/min, and determined wavelength is 450nm;
D) sampling volume: 300 μ L;
Preparing liquid 150mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthol, concentrated solution being carried out lyophilize, obtains fucoxanthol 27mg, purity 99.03%.
Embodiment 8
1) enzyme digestion reaction: get fucoxanthine crude product (purity is 5%) 1000mg, add lipase 1000mg, Taurocholic acid sodium salt 1000mg, be dissolved in the 0.1M PBS buffered soln of pH=7, react 2h at 37 DEG C after, be concentrated into dry, add the methyl alcohol supersound extraction of 80mL, filter, filtrate is concentrated into dry, obtains fucoxanthol reactant 498mg;
2) initial gross separation purifying: adopt silica gel column chromatography, use sherwood oil-acetone (20:1-3:1) to carry out wash-out respectively, collects (25:1 and 10:1) elution fraction, concentrate drying, obtains fucoxanthol crude product 79mg, content 41%;
3) fucoxanthol is refined:
A) configuration of raw material: solution fucoxanthol crude product being configured to 6mg/mL;
B) instrument: preparative high performance liquid chromatography;
C) chromatographic condition: half preparative chromatography post is C8 post (250mm × 10mm), and flow phase system is that 70% methyl alcohol is water-soluble
Liquid, flow rate of mobile phase is 6mL/min, and determined wavelength is 450nm;
D) sampling volume: 300 μ L;
Preparing liquid 110mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthol, concentrated solution being carried out lyophilize, obtains fucoxanthol 23mg, purity 99.00%.
Embodiment 9
1) enzyme digestion reaction: get fucoxanthine crude product (purity is 1%) 1000mg, add lipase 800mg, Taurocholic acid sodium salt 800mg (i.e. raw material: lipase: catalyzer=1:40:40), be dissolved in the 0.1M PBS buffered soln of pH=7, react 2h at 37 DEG C after, be concentrated into dry, add the methyl alcohol supersound extraction of 80mL, filter, filtrate is concentrated into dry, obtains fucoxanthol reactant 367mg;
2) initial gross separation purifying: adopt silica gel column chromatography, use sherwood oil-acetone (20:1 ~ 3:1) to carry out wash-out respectively, collects (25:1 and 10:1) elution fraction, concentrate drying, obtains fucoxanthol crude product 53mg, content 28%;
3) fucoxanthol is refined:
A) configuration of raw material: solution fucoxanthol crude product being configured to 6mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C8 post (250mm × 10mm), and flow phase system is 90% methanol aqueous solution, and flow rate of mobile phase is 6mL/min, and determined wavelength is 450nm;
D) sampling volume: 300 μ L;
Preparing liquid 55mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthol, concentrated solution being carried out lyophilize, obtains fucoxanthol 9.8mg, purity 99.03%.
Embodiment 10
1) enzyme digestion reaction: get fucoxanthine crude product (purity is 50%) 100mg, add lipase 200mg, Taurocholic acid sodium salt 200mg, be dissolved in the 0.1M PBS buffered soln of pH=7, react 2h at 37 DEG C after, be concentrated into dry, add the methyl alcohol supersound extraction of 80mL, filter, filtrate is concentrated into dry, obtains fucoxanthol reactant 332mg;
2) initial gross separation purifying: adopt silica gel column chromatography, use petroleum ether-ethyl acetate (20:1 ~ 3:1) to carry out wash-out respectively, collect (25:1 and 10:1) elution fraction, concentrate drying, obtain fucoxanthol crude product 43mg, content 78%;
3) fucoxanthol is refined:
A) configuration of raw material: solution fucoxanthol crude product being configured to 6mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C18 post (250mm × 10mm), and flow phase system is 60% aqueous ethanolic solution, and flow rate of mobile phase is 6mL/min, and determined wavelength is 450nm;
D) sampling volume: 300 μ L;
Preparing liquid 95mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthol, concentrated solution being carried out lyophilize, obtains fucoxanthol 28.8mg, purity 99.13%.
Embodiment 11
1) enzyme digestion reaction: get fucoxanthine crude product (purity is 50%) 100mg, add lipase 200mg, Taurocholic acid sodium salt 200mg, be dissolved in the 0.1M PBS buffered soln of pH=7, react 2h at 37 DEG C after, be concentrated into dry, add the methyl alcohol supersound extraction of 80mL, filter, filtrate is concentrated into dry, obtains fucoxanthol reactant 350mg;
2) initial gross separation purifying: adopt silica gel column chromatography, use petroleum ether-ethyl acetate (20:1 ~ 3:1) to carry out wash-out respectively, collect (25:1 and 10:1) elution fraction, concentrate drying, obtain fucoxanthol crude product 53mg, content 73%;
3) fucoxanthol is refining
A) configuration of raw material: solution fucoxanthol crude product being configured to 6mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C18 post (250mm × 10mm), and flow phase system is 95% aqueous ethanolic solution, and flow rate of mobile phase is 6mL/min, and determined wavelength is 450nm;
D) sampling volume: 300 μ L;
Preparing liquid 75mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthol, concentrated solution being carried out lyophilize, obtains fucoxanthol 27mg, purity 99.01%.
Embodiment 12
1) enzyme digestion reaction: get fucoxanthine crude product (purity is 95%) 100mg, add lipase 300mg, Taurocholic acid sodium salt 300mg, be dissolved in the 0.1M PBS buffered soln of pH=7, react 2h at 37 DEG C after, be concentrated into dry, add the methyl alcohol supersound extraction of 80mL, filter, filtrate is concentrated into dry, obtains fucoxanthol reactant 350mg;
2) fucoxanthol is refined:
A) configuration of raw material: solution fucoxanthol crude product being configured to 10mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C18 post (250mm × 10mm), and flow phase system is 95% acetonitrile solution, and flow rate of mobile phase is 6mL/min, and determined wavelength is 450nm;
D) sampling volume: 600 μ L;
Preparing liquid 135mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthol, concentrated solution being carried out lyophilize, obtains fucoxanthol 59mg, purity 99.01%.
Embodiment 13
1) enzyme digestion reaction: get fucoxanthine crude product (purity is 95%) 100mg, add lipase 300mg, Taurocholic acid sodium salt 300mg, be dissolved in the 0.1M PBS buffered soln of pH=7, react 2h at 37 DEG C after, be concentrated into dry, add the methyl alcohol supersound extraction of 80mL, filter, filtrate is concentrated into dry, obtains fucoxanthol reactant 450mg;
2) fucoxanthol is refined:
A) configuration of raw material: solution fucoxanthol crude product being configured to 10mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: half preparative chromatography post is C18 post (250mm × 10mm), and flow phase system is 75% acetonitrile solution, and flow rate of mobile phase is 6mL/min, and determined wavelength is 450nm;
D) sampling volume: 600 μ L;
Preparing liquid 235mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthol, concentrated solution being carried out lyophilize, obtains fucoxanthol 62mg, purity 99.09%.
Embodiment 14
1) enzyme digestion reaction: get fucoxanthine crude product (purity is 95%) 500mg, add lipase 1500mg, Taurocholic acid sodium salt 1500mg, be dissolved in the 0.1M PBS buffered soln of pH=7, react 2h at 37 DEG C after, be concentrated into dry, add the methyl alcohol supersound extraction of 100mL, filter, filtrate is concentrated into dry, obtains fucoxanthol reactant 980mg;
2) fucoxanthol is refined:
A) configuration of raw material: solution fucoxanthol crude product being configured to 15mg/mL;
B) instrument: preparative high performance liquid chromatography;
C) chromatographic condition: preparative chromatography post is C18 post (250mm × 20mm), and flow phase system is 90% methanol aqueous solution, and flow rate of mobile phase is 28mL/min, and determined wavelength is 450nm;
D) sampling volume: 1000 μ L;
Preparing liquid 690mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthol, concentrated solution being carried out lyophilize, obtains fucoxanthol 435mg, purity 99.19%.Embodiment 15
1) enzyme digestion reaction: get fucoxanthine crude product (purity is 95%) 2.0g, add lipase 10.0g, Taurocholic acid sodium salt 5.0g, be dissolved in the 0.1M PBS buffered soln of pH=7, react 2h at 37 DEG C after, be concentrated into dry, add the methyl alcohol supersound extraction of 200mL, filter, filtrate is concentrated into dry, obtains fucoxanthol reactant 3.9g;
2) fucoxanthol is refined:
A) configuration of raw material: solution fucoxanthol crude product being configured to 40mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: preparative chromatography post is C18 post (250mm × 50mm), and flow phase system is 90% methanol aqueous solution, and flow rate of mobile phase is 80mL/min, and determined wavelength is 450nm;
D) sampling volume: 2000 μ L;
Preparing liquid 3500mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthol, concentrated solution being carried out lyophilize, obtains fucoxanthol 1.68g, purity 99.29%.Embodiment 16
1) enzyme digestion reaction: get fucoxanthine crude product (purity is 95%) 10.0g, add lipase 40.0g, Taurocholic acid sodium salt 20.0g, be dissolved in the 0.1M PBS buffered soln of pH=7, react 2h at 37 DEG C after, be concentrated into dry, add the methyl alcohol supersound extraction of 500mL, filter, filtrate is concentrated into dry, obtains fucoxanthol reactant 23.9g;
2) fucoxanthol is refined:
A) configuration of raw material: solution fucoxanthol crude product being configured to 40mg/mL;
B) instrument: Semipreparative chromatography;
C) chromatographic condition: preparative chromatography post is C18 post (250mm × 50mm), and flow phase system is 90% methanol aqueous solution, and flow rate of mobile phase is 200mL/min, and determined wavelength is 450nm;
D) sampling volume: 5000 μ L;
Preparing liquid 9500mL, concentrating under reduced pressure at 30 DEG C by running preparative high performance liquid chromatography acquisition fucoxanthol, concentrated solution being carried out lyophilize, obtains fucoxanthol 7.68g, purity 99.29%.
Claims (10)
1. a preparation method for fucoxanthol, is characterized in that comprising the following steps;
(1), with fucoxanthine high-purity monomer or with fucoxanthine extract for raw material;
(2), by raw material and lipase, catalyst mix, react in water-bath with after PBS buffer solution, obtain reaction solution.
2. the preparation method of fucoxanthol as claimed in claim 1; wherein said catalyzer is lipase accelerator, the Sodium cholic acid salts substances such as preferred sodium deoxycholate, Taurocholic acid sodium salt, Sodium cholic acid, NaGC, deoxidation Taurocholic acid sodium salt, Taurocholic acid sodium salt salt hydrate, sweet ammonia SODIUM CHENODIOL, gallodesoxycholic acid sodium, sodium taurochenodeoxycholate.
3. the preparation method of fucoxanthol as claimed in claim 1, is characterized in that step 2) described in by raw material and lipase, catalyst mix, raw material: lipase: the ratio of catalyzer is 1:1:1 to 1:40:40.
4. the preparation method of fucoxanthol as claimed in claim 1, is characterized in that step 2) described in the pH value of PBS damping fluid be 3-11, the temperature of water-bath is 20-70 DEG C, and the reaction times is 0-24h.
5. the preparation method of fucoxanthol according to claim 1, characterized by further comprising step 3), by step 2) reaction solution that obtains carries out being concentrated into dry, use methyl alcohol supersound extraction, extracting solution is concentrated into dry, obtains fucoxanthol crude extract.
6. the preparation method of fucoxanthol as claimed in claim 5, it is characterized in that also selectablely comprising step 4), by step 3) crude extract that obtains adopts column chromatography to carry out initial gross separation, collects fucoxanthol cut, concentratedly obtains fucoxanthol coarse raw materials.
7. the preparation method of the fucoxanthol as described in one of claim 5-6, characterized by further comprising step 5), by step 3) the fucoxanthol crude extract that obtains or step 4) the fucoxanthol coarse raw materials moving phase that obtains dissolves, proceed in half preparation/preparative high performance liquid chromatography sample injection bottle, start half preparation/preparative high performance liquid chromatography and carry out separation and purification, trigger run tank by on-line ultraviolet detection signal and automatically collect fucoxanthol refined solution.
8. the preparation method of fucoxanthol as claimed in claim 7, characterized by further comprising step 6), by step 5) in fucoxanthol refined solution carry out concentrating under reduced pressure after, get concentrated solution and carry out lyophilize, obtain high-purity fucoxanthol.
9. the preparation method of fucoxanthol as claimed in claim 7, it is characterized in that the moving phase of half described preparation/preparative high performance liquid chromatography employing is the one in methanol aqueous solution, aqueous ethanolic solution or acetonitrile solution, the concentration of volume percent of methanol aqueous solution is 70-95%, the volume percent of aqueous ethanolic solution is 60-95%, and the volume percent of acetonitrile solution is 55-95%.
10. the preparation method of fucoxanthol as claimed in claim 6, is characterized in that wherein step 4) described in column chromatography be silica gel column chromatography or macroporous adsorbent resin column chromatography.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510147401.4A CN104789611A (en) | 2015-03-31 | 2015-03-31 | Preparation method of fucoxanthol |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510147401.4A CN104789611A (en) | 2015-03-31 | 2015-03-31 | Preparation method of fucoxanthol |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104789611A true CN104789611A (en) | 2015-07-22 |
Family
ID=53554803
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510147401.4A Pending CN104789611A (en) | 2015-03-31 | 2015-03-31 | Preparation method of fucoxanthol |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104789611A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106749110A (en) * | 2016-12-29 | 2017-05-31 | 国家***第三海洋研究所 | A kind of method that use reducing agent prepares fucoxanthol |
| CN109757723A (en) * | 2019-01-25 | 2019-05-17 | 集美大学 | A kind of eyesight protection function food and preparation method thereof containing fucoxanthol |
| CN112341514A (en) * | 2019-08-06 | 2021-02-09 | 杜心赟 | Deoxycholic acid compound, pharmaceutical composition and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102040569A (en) * | 2009-10-20 | 2011-05-04 | 北京绿色金可生物技术股份有限公司 | Carotinoid derivatives and preparation method and application thereof |
-
2015
- 2015-03-31 CN CN201510147401.4A patent/CN104789611A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102040569A (en) * | 2009-10-20 | 2011-05-04 | 北京绿色金可生物技术股份有限公司 | Carotinoid derivatives and preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| AKIRA ASAI ET AL.: "BIOTRANSFORMATION OF FUCOXANTHINOL INTO AMAROUCIAXANTHIN A IN MICE AND HEPG2 CELLS: FORMATION AND CYTOTOXICITY OF FUCOXANTHIN METABOLITES.", 《DRUG METABOLISM AND DISPOSITION》 * |
| HAYATO MAEDA ET AL.: "Fucoxanthin and its metabolite, fucoxanthinol，suppress adipocyte differentiation in 3T3-L1 cells.", 《INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE》 * |
| TATSUYA SUGAWARA ET AL.: "Antiangiogenic Activity of Brown Algae Fucoxanthin and Its Deacetylated Product, Fucoxanthinol.", 《J. AGRIC. FOOD CHEM.》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106749110A (en) * | 2016-12-29 | 2017-05-31 | 国家***第三海洋研究所 | A kind of method that use reducing agent prepares fucoxanthol |
| CN106749110B (en) * | 2016-12-29 | 2019-04-02 | 国家***第三海洋研究所 | A method of fucoxanthol is prepared using reducing agent |
| CN109757723A (en) * | 2019-01-25 | 2019-05-17 | 集美大学 | A kind of eyesight protection function food and preparation method thereof containing fucoxanthol |
| CN112341514A (en) * | 2019-08-06 | 2021-02-09 | 杜心赟 | Deoxycholic acid compound, pharmaceutical composition and application thereof |
| WO2021023100A1 (en) * | 2019-08-06 | 2021-02-11 | 杜心赟 | Deoxycholic acid compounds, pharmaceutical compositions and uses thereof |
| CN112341514B (en) * | 2019-08-06 | 2023-11-21 | 杜心赟 | Deoxycholic acid compound, pharmaceutical composition and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Usuki et al. | Extraction and isolation of shikimic acid from Ginkgo biloba leaves utilizing an ionic liquid that dissolves cellulose | |
| CN103012327B (en) | Preparation method of fucoxanthin | |
| CN103193735B (en) | Extraction method for taxus chinensis taxol activity extract | |
| CN103450120A (en) | Preparation method for extracting taxol from taxus chinensis | |
| Kim et al. | Enhancement of extraction efficiency of paclitaxel from biomass using ionic liquid-methanol co-solvents under acidic conditions | |
| CN104789611A (en) | Preparation method of fucoxanthol | |
| CN110563781B (en) | Preparation method of albizzia bark neolignan monomer compound | |
| CN101230053B (en) | Method for ultrasonic extraction and separation of 10-deacetyl baccatin III from yew branch leaves | |
| CN108299453B (en) | Method for separating psoralen, isopsoralen and bakuchiol from fructus psoraleae | |
| CN101921277B (en) | Method for simultaneously preparing vasicine and vasicinone from peganum harmala | |
| CN106561722A (en) | Algal inhibition application of undaria pinnatifida and separation and purification method of undaria pinnatifida algal inhibition active substance | |
| CN103265536A (en) | Preparation method of swertisin | |
| CN106226426A (en) | A kind of high performance liquid chromatography splits the method for canagliflozin five-membered ring impurity enantiomer | |
| CN103265548A (en) | Extraction method of dendrobine | |
| CN103570647A (en) | Method for preparing high-purity paclitaxel from taxus chinensis cell culture fluid | |
| CN101531590A (en) | Ginkgoic acid preparation method using macroporous adsorption resin | |
| CN102241686A (en) | Preparation method of karanjin | |
| CN104262231A (en) | Method for extracting and separating L-tryptophan from nitraria tangutorum bobr seeds | |
| CN104031008B (en) | A kind of preparation method of taxol crude extract | |
| CN103588736B (en) | 13-acetyl-9-dihydrobaccatin III extraction separation method | |
| CN103910783B (en) | A kind of preparation method of high-purity echinocandin B parent nucleus | |
| CN106749110B (en) | A method of fucoxanthol is prepared using reducing agent | |
| CN102391328B (en) | Method for simultaneously preparing chemical reference substances magnoloside A and magnoloside B | |
| CN104447787A (en) | Method for separating and purifying two sesquiterpene lactone compounds from ambrosia artemisiifolia | |
| CN104610209A (en) | Industrial method for improving purity of barbaloin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150722 |