CN1233616C - Methyl eicosapentaenoic acid preparing and purifying method from pavlova viridis - Google Patents
Methyl eicosapentaenoic acid preparing and purifying method from pavlova viridis Download PDFInfo
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- CN1233616C CN1233616C CN 200310106399 CN200310106399A CN1233616C CN 1233616 C CN1233616 C CN 1233616C CN 200310106399 CN200310106399 CN 200310106399 CN 200310106399 A CN200310106399 A CN 200310106399A CN 1233616 C CN1233616 C CN 1233616C
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- eicosapentaenoic acid
- acetone
- methyl ester
- acid methyl
- hexane
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Abstract
The present invention relates to a method for preparing and purifying eicosapentaenoic acid methyl ester from pavlova viridis. Freeze-dried pavlova viridis powder of is added in chloroacetyl-methanol with the concentration of 5%, sealing is carried out after nitrogen is filled, reaction is carried out for 0.5 to 2 hours at 80 to 100 DEG C, the pavlova viridis powder is cooled to room temperature, redistilled water with the same volume and n-hexane are respectively added, oscillation and extraction are carried out, extraction is carried out for a plurality of times, n-hexane is evaporated under the protection of nitrogen after extract is merged, fatty acid methyl ester is obtained, silver silica gel columns are coated for chromatography, gradient elution is carried out with propanone-n-hexane solution, eluent with the purity of eicosapentaenoic acid methyl ester above 95% is merged, propanone and n-hexane are evaporated under the protection of nitrogen, eicosapentaenoic acid methyl ester is obtained, and purity is above 95%. The method of the present invention has the advantages of simplicity, easy operation, low cost and high purity of eicosapentaenoic acid methyl ester.
Description
Technical field
The present invention relates to Ba Fuzao and timnodonic acid, specifically, relate to prepare the method for methyl eicosapentaenoic acid from crust husband algae.
Background technology
Pavlova viridis (Pavlova viridis) is a kind of yellowish green small chrysophyceae.Frond ovalize or circle, acellular wall.The about 4-6 μ of this frustule diameter m can move, and is that a kind of comfort zone is wide, and illumination requires the low little algae of single-cell sea.The good bait of the Chang Zuowei shrimps young and economic shellfish.The pavlova viridis that studies show that in recent years is rich in timnodonic acid polyunsaturated fatty acids such as (Eicosapentaenoic acid call EPA in the following text), and its EPA content accounts for the 12-15% (changing according to the culture condition difference) of total fat.
Polyunsaturated fatty acids such as EPA are present in the alga cells glycolipid, and obtain methyl eicosapentaenoic acid generally at first needs to extract total ester from crust husband algae, and saponification makes it into free acid, carries out esterification then, and separation, purifying obtain methyl eicosapentaenoic acid at last.Separation, purifying mainly contain low-temperature freezing, urea adduct method, molecular distillation method, supercritical CO
2Extraction process etc.But yield and the purity of these methods or EPA are lower, and perhaps cost is too high.
Summary of the invention
The purpose of this invention is to provide the method that a kind of step is simple, cost is low, purity is high, yield is high from pavlova viridis preparation and purifying methyl eicosapentaenoic acid.
Technical scheme of the present invention is as follows:
A kind of method from pavlova viridis preparation and purifying methyl eicosapentaenoic acid, it comprises the following steps:
Step 1. is pavlova viridis freeze-dried algae powder N gram, in the container of packing into, add 20N～30N milliliter 5% chloracetyl-methyl alcohol (promptly 1: 20v/v) airtight behind the inflated with nitrogen, reacted 0.5～2 hour down in 80-100 ℃, be cooled to room temperature,
Step 2. adds the double distilled water and the normal hexane of equal volume respectively in the reaction mixture of step 1 gained, oscillation extraction, draw supernatant liquid after, add the normal hexane re-extract of equivalent again, no longer turn to be yellow to supernatant liquor,
Behind step 3. combining extraction liquid, use the siccative drying, the elimination siccative boils off normal hexane under nitrogen protection, obtains fatty acid methyl ester 0.046N～0.07N gram,
Step 4. is carried out chromatography with the fatty acid methyl ester that step 3 obtains with being coated with silver-colored silicagel column, with acetone-hexane solution gradient elution, collects elutriant respectively, and merging contains methyl eicosapentaenoic acid purity and is higher than 95% elutriant,
In the above-mentioned preparation and the step 4 of purification process, acetone-hexane solution gradient elution can be respectively is acetone-hexane solution of 0.5%, 1%, 5%, 10% and 15% wash-out successively with the acetone concentration expressed in percentage by volume.
In the above-mentioned preparation and the step 4 of purification process, being coated with the silver-colored silica gel of being coated with of silver-colored silicagel column can be prepared as follows:
A certain amount of silica gel for chromatography is added in the two volumes dehydrated alcohol, mix into suspension.Silver Nitrate is dissolved in an amount of 70% ethanol, and it is even to add above-mentioned chromatographic silica gel thorough mixing.The weight ratio of Silver Nitrate and silica gel is 1: 10.Boil off ethanol then, the silver-colored silica gel of being coated with of gained is placed baking oven,, after the cooling, preserve in the dark place in 120 ℃ of high-temperature activations (more than 12 hours).
Method steps from pavlova viridis preparation and purifying methyl eicosapentaenoic acid of the present invention is simple, consumes and lacks, and cost is low, the yield height, and yield can reach 65～72% of theoretical value, the purity height, purity can reach more than 95%.
Description of drawings
Fig. 1 is coated with silver-colored silica gel column chromatography-gradient concentration elutriant among the embodiment 2, the gas chromatogram of 15% acetone-normal hexane first pipe elutriant, and wherein the retention time and the peak area at a peak are as follows:
Peak?RetTime?Type Width Area Height Area
# [min] [min] [pA*s] [pA] ％
----|-------|----|-------|----------|----------|--------|
1 14.148?PB 0.0681 4.08046?7.91750e-1 0.42113
2 15.930?PP 0.0667 7.70089 1.64221 0.79478
3 18.151?PB 0.0636 923.32898 187.03178?95.29351
4 18.589?PB 0.0624 3.55124?6.86098e-1 0.36651
5 19.541?BB 0.0718 10.97733 1.88797 1.13293
6 21.027?BB 0.0713 14.81180 2.86513 1.52867
7 21.685?PV 0.0640 2.02365?3.86956e-1 0.20885
8 22.399?PV 0.0702 2.45727?4.26771e-1 0.25361
Totals： 968.93161 195.71867
Fig. 2 is coated with silver-colored silica gel column chromatography-gradient concentration elutriant among the embodiment 2, the gas chromatogram of 15% acetone-normal hexane second pipe elutriant, and wherein the retention time and the peak area at a peak are as follows:
Peak?RetTime?Type Width Area Height Area
# [min] [min] [pA*s] [pA] ％
----|-------|----|-------|----------|----------|--------|
1 14.155?PB 0.0659 2.07125?3.90620e-1 0.38716
2 15.945?BB 0.0682 4.44554?9.56831e-1 0.83097
3 18.143?PB 0.0714 517.56049 105.23692?96.74304
4 19.557?BP 0.0857 6.76244 1.01491 1.26404
5 21.044?PB 0.0622 1.90134?3.80979e-1 0.35540
6 22.405?BB 0.0742 2.24367?3.67851e-1 0.41939
Totals： 534.98472 108.34811
Results?obtained?with?enhanced?integrator！
Fig. 3 is coated with silver-colored silica gel column chromatography-gradient concentration elutriant among the embodiment 2, the gas chromatogram of 15% acetone-normal hexane the 3rd pipe elutriant, and wherein the retention time and the peak area at a peak are as follows:
Peak?RetTime?Type Width Area Height Area
# [min] [min] [pA*s] [pA] ％
----|-------|----|-------|----------|----------|--------|
1 15.952?BP 0.0648 2.47534?5.27118e-1 0.86594
2 18.134?BB 0.0774 278.85254 55.68338?97.54986
3 19.563?BB 0.0858 4.52853?6.54070e-1 1.58420
Totals： 285.85641 56.86457
Results?obtained?with?enhanced?integrator！
Fig. 4 is coated with silver-colored silica gel column chromatography-gradient concentration elutriant among the embodiment 2, the gas chromatogram of 15% acetone-normal hexane the 4th and the 5th pipe elutriant, and wherein the retention time and the peak area at a peak are as follows:
Peak?RetTime?Type Width Area Height Area
# [min] [min] [pA*s] [pA] ％
----|-------|----|-------|----------|----------|--------|
1 18.129?PB 0.0772 136.59071 27.39506?97.74114
2 19.570?BP 0.1351 3.15670?2.83150e-1 2.25886
Totals： 139.74741 27.67821
Results?obtained?with?enhanced?integrator！
Embodiment
Embodiment 1. algae culture and results
The little algae pavlova viridis of single-cell sea is cultivated through this laboratory rejuvenation available from Chinese Academy of Sciences marine laboratory, Qingdao, is stored in 21-27 ℃.Culture condition is as follows: 6000-9000lux, 12:12L/D, aerated culture.Exponential growth results in latter stage were collected frond in the centrifugal 8-15 of 4000-8000g minute, and distilled water wash is to remove salt and other impurity, lyophilize, cryogenic freezing preservation.
Embodiment 2. is coated with the preparation of silver-colored silicagel column:
A certain amount of silica gel for chromatography is added in the two volumes dehydrated alcohol, mix into suspension.Silver Nitrate is dissolved in an amount of 70% ethanol, and it is even to add above-mentioned chromatographic silica gel thorough mixing.The weight ratio of Silver Nitrate and silica gel is 1: 10.Boil off ethanol then, the silver-colored silica gel of being coated with of gained is placed baking oven,, after the cooling, preserve in the dark place in 120 ℃ of high-temperature activations (more than the 12h).
Embodiment 3. is from pavlova viridis preparation and purifying methyl eicosapentaenoic acid
From pavlova viridis preparation and purifying methyl eicosapentaenoic acid, it comprises the following steps:
Step 1. in the screw-cap test tube of packing into, adds 20 milliliters of 5% chloracetyl-methyl alcohol (promptly 1: 20 v/v) with pavlova viridis freeze-dried algae powder 1.0 grams, and is airtight behind the inflated with nitrogen, reacts 2 hours down in 80 ℃, is cooled to room temperature,
Step 2. adds the double distilled water and the normal hexane of equal volume respectively in the reaction mixture of step 1 gained, oscillation extraction, draw supernatant liquid after, add the normal hexane re-extract of equivalent again, no longer turn to be yellow to supernatant liquor,
Behind step 3. combining extraction liquid, use the siccative drying, the elimination siccative boils off normal hexane under nitrogen protection, obtains fatty acid methyl ester 0.046 gram,
Step 4. is carried out chromatography with the fatty acid methyl ester that step 3 obtains with being coated with silver-colored silicagel column, be 0.5% with acetone concentration respectively, 1%, 5%, each 25ml of the acetone-hexane solution of 10%, 15% (v/v) is by concentration wash-out successively from low to high, and flow rate control is at 1.3～2.0ml/min, every 5ml collects a pipe, collects the effluent liquid of each different concns gradient respectively and does the GC analysis.Find when the acetone concentration of acetone-normal hexane elutriant is lower than 10%, some saturated and low fatty acid methyl ester and other impurity of degree of unsaturation can be eluted substantially fully, EPA begins wash-out and comes out in last when pipe of 10% acetone-normal hexane, when the elutriant acetone concentration reaches 15% (v/v), obtaining purity successively is 95.29%, 96.74%, the EPA methyl esters (seeing gas chromatogram, Fig. 1-4) of 97.55%, 97.74% (the 4th and the 5th pipe elutriant of merging).The elutriant that merges 15% acetone-normal hexane can obtain purity greater than 95% EPA methyl esters, and yield is about 60%.
Embodiment 4. is from pavlova viridis preparation and purifying methyl eicosapentaenoic acid
From pavlova viridis preparation and purifying methyl eicosapentaenoic acid, it comprises the following steps:
Step 1. in the screw-cap test tube of packing into, adds 30 milliliters of 5% chloracetyl-methyl alcohol (promptly 1: 20 v/v) with pavlova viridis freeze-dried algae powder 1.0 grams, and is airtight behind the inflated with nitrogen, reacts 0.5 hour down in 100 ℃, is cooled to room temperature,
Step 2. adds the double distilled water and the normal hexane of equal volume respectively in the reaction mixture of step 1 gained, oscillation extraction, draw supernatant liquid after, add the normal hexane re-extract of equivalent again, no longer turn to be yellow to supernatant liquor,
Behind step 3. combining extraction liquid, use the siccative drying, the elimination siccative boils off normal hexane under nitrogen protection, obtains fatty acid methyl ester 0.07 gram,
Step 4. is carried out chromatography with the fatty acid methyl ester that step 3 obtains with being coated with silver-colored silicagel column, with acetone-hexane solution gradient elution, collects elutriant with embodiment respectively, and merging contains methyl eicosapentaenoic acid purity and is higher than 95% elutriant,
Claims (2)
1. the method from pavlova viridis preparation and purifying methyl eicosapentaenoic acid is characterized in that it comprises the following steps:
Step 1. in the container of packing into, adds pavlova viridis freeze-dried algae powder 1N gram 20N～30N ml volumes percentage concentration and is chloracetyl-methyl alcohol of 5%, and is airtight behind the inflated with nitrogen, reacts 0.5～2 hour down in 80-100 ℃, is cooled to room temperature,
Step 2. adds the double distilled water and the normal hexane of equal volume respectively in the reaction mixture of step 1 gained, oscillation extraction, draw supernatant liquid after, add the normal hexane re-extract of equivalent again, no longer turn to be yellow to supernatant liquor,
Behind step 3. combining extraction liquid, use the siccative drying, the elimination siccative boils off normal hexane under nitrogen protection, obtain fatty acid methyl ester,
Step 4. is carried out chromatography with the fatty acid methyl ester that step 3 obtains with being coated with silver-colored silicagel column, with acetone-hexane solution gradient elution, collects elutriant respectively, and merging contains methyl eicosapentaenoic acid purity and is higher than 95% elutriant,
Step 5. boils off acetone and normal hexane under nitrogen protection, promptly get methyl eicosapentaenoic acid, and purity is higher than 95%.
2. preparation according to claim 1 and purification process is characterized in that: in step 4, acetone-hexane solution gradient elution is acetone-hexane solution of 0.5%, 1%, 5%, 10% and 15% wash-out successively with the acetone concentration expressed in percentage by volume respectively.
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| TWI414362B (en) | 2011-05-18 | 2013-11-11 | Ind Tech Res Inst | Extraction apparatus |
| CN102517156A (en) * | 2011-12-31 | 2012-06-27 | 中国海洋大学 | High-efficiency quick preparation method of microalgae fatty acid methyl ester |
| CN104195148B (en) * | 2014-09-16 | 2016-08-10 | 合肥工业大学 | A kind of pavlova viridis delta-5 delta 8 desaturase genes and preparation method for EPA synthesis |
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