CN102911201B - A kind of technique preparing standard of phosphatidylcholine in Euphausia superba - Google Patents
A kind of technique preparing standard of phosphatidylcholine in Euphausia superba Download PDFInfo
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Abstract
The invention discloses a kind of technique preparing standard of phosphatidylcholine in Euphausia superba, step is as follows: (1) gets dry krill, adds normal hexane, and stir extraction 3 times, united extraction liquid, rotary evaporation, obtains shrimp sauce; (2) in shrimp sauce, add acetone, stir extraction 6 times, merging filtrate, rotary evaporation, must containing the oily sample liquid of phosphatide; (3) preparative chromatography post; (4) get the oily sample liquid 1.5 ~ 3ml of step (2) gained containing phosphatide, slowly join in chromatography column, use chloroform-methanol elution, collect elutriant; (5) concentrated by each part elutriant collected, each part eluant component of thin-layer chromatography Qualitative Identification, merges the concentrated solution of single for tool phosphatidyl choline component, evaporation of solvent, obtain phosphatidylcholine standard substance, detect through high pressure liquid phase analysis, PC purity >=98%.The present invention has prepared standard of phosphatidylcholine in Euphausia superba, for contribution has been made in the detection of following krill deep processed product.
Description
Technical field
The present invention relates to a kind of technique preparing standard of phosphatidylcholine in Euphausia superba.
Background technology
Krill (Euphausiasuperba) has another name called large krill or Antarctic krill, is the krill of one way of life in the waters, the Antarctica of Antarctic Ocean.Krill aboundresources, the highest according to estimates have several hundred million ton catchability, is more than 1 times of the existing fishery production in the world, has huge development and utilization potentiality.Main fishery country of the world is Japanese and Polish.Product is mainly used as cooking in Japan, and elsewhere is then as animal-feed and bait in the world.Under the background that world's marine fishery resources generally fails, krill resource is day by day subject to countries in the world and pays close attention to.
It is reported, containing abundant phospholipid composition in krill, phosphatidylcholine (the phosphatidylcholine being called as " lipotropic factor " is rich in phosphatide, abbreviation PC, also known as Yelkin TTS), it is effective with the cerebrovascular, the microvascular disease such as cardiovascular to fatty liver, lipodystrophy, arteriosclerosis, tetter; Hypertension, treatment dry skin tinea can also be prevented.Phosphatidylcholine can directly use as injection, the emulsifying agent of fat transfusion especially.Phosphatidylcholine also can be used as liposome, and medicine as stronger in some toxicity, as the bag loaded body of cancer therapy drug cis-platinum, can make cancer therapy drug smoothly by the cytolemma using phosphatide as composition.Namely the precious medicine " essentiale " of China's treatment hepatopathy is take phosphatidylcholine as main component.In addition, phosphatidylcholine is at high grade paint, and the aspects such as superior cosmetics, agricultural chemicals, sterilant, textiles, petroleum product (as rubber), electronic industry also have different excellent purposes.
China's krill has now entered the expedition stage, starts to fish in a large number after about 1 ~ 2 year, and the subject matter faced when the time comes is the deep processing and utilization of krill.Mainly prepared by euphausia superba sauce to the processing of krill abroad, wherein phospholipids content accounts for 40%, phosphatidylcholine is the main component of shrimp sauce phosphatide, shrimp sauce likely becomes the raw material of best phosphatidylcholine processing, production euphausia superba sauce, production Antarctic krill phosphatidyl choline, all needs the content of qualitative and quantitative detection PC.The standardization of krill PC is needed in testing process.Preparing krill PC standard substance is keys that following krill deep processed product detects.PC standard substance itself are also a kind of products.
Summary of the invention
For above-mentioned prior art, the invention provides a kind of technique preparing standard of phosphatidylcholine in Euphausia superba.
The present invention is achieved by the following technical solutions:
Prepare a technique for standard of phosphatidylcholine in Euphausia superba, step is as follows:
(1) get dry krill (water content is 8% ~ 10%) 100g, add the normal hexane of 500 ~ 600ml, stir extraction at 20 ~ 35 DEG C 3 times, extract 1h at every turn, often extract once, filter and dry krill is leached; United extraction liquid (about 1350 ~ 1700ml), at 30 ~ 45 DEG C, rotary evaporation under-0.07 ~-0.09Mpa, obtains shrimp sauce (about 4.5 ~ 5.5g);
(2) in the shrimp sauce of step (1) gained, add the acetone of 5 ~ 7 times (refer to quality volume multiple, namely 1g material adds 5 ~ 7ml liquid, lower same), stir extraction at 20 ~ 35 DEG C 6 times, extract 0.5h, extracting liquid filtering at every turn, often extract once, filter to get filtrate and filter residue; Merge the filtrate (about 135 ~ 210ml) of 6 times, at 30 ~ 40 DEG C, rotary evaporation under-0.07 ~-0.09Mpa, must containing the oily sample liquid of phosphatide;
(3) preparation of chromatography column: take 65 ~ 75g silica gel, activates 6 ~ 10h at 110 ~ 120 DEG C; In silica gel, add 3 times of chloroforms (quality volume multiple), stir into pulpous state; Adopt wet method dress post method that silica gel is encased in 3 × 30cm
2in chromatography column, leave standstill, make silica gel fully be settled down to volume stability constant;
(4) get step (2) gained containing the oily sample liquid 1.5 ~ 3ml of phosphatide, slowly join in chromatography column, with chloroform: methyl alcohol=1:1 ~ 2(volume ratio) elution, flow velocity is 2 ~ 3ml/min, collects elutriant by every part of 100 ~ 300ml;
(5) concentrated by each part elutriant collected, each part eluant component of thin-layer chromatography Qualitative Identification, merges the concentrated solution of single for tool phosphatidyl choline component, evaporation of solvent, obtain phosphatidylcholine standard substance (about 8 ~ 12mg), detect through HPLC, purity >=98% of phosphatidylcholine.
The thin-layer chromatography qualitative checking method of described Antarctic krill phosphatidyl choline (PC) is:
Thin-layer chromatography uses GF254 silica gel thin-layer plate, developping agent is chloroform: methyl alcohol: water: ethamine=14:6:0.6:5(V:V:V:V), exhibition, apart from being 6cm, gets the concentrated solution point sample of each elutriant, exhibition is dried after finishing, and puts into dibromothymolsulfonphthalein dye liquor cylinder dyeing 12 seconds; Dry after taking-up, dried thin layer plate is placed on thin layer chromatography scanner and observes: the Rf value of PC is 0.37.
The HPLC quantitative detecting method of described Antarctic krill phosphatidyl choline (PC) is:
Adopt the U.S. (waters) HPLC instrument, 2489 UV-detector, SepaxHP-silica(4.6 × 250mm5um) chromatographic column, moving phase is made, flow velocity 1ml/min, column temperature 35 DEG C with methyl alcohol, determined wavelength 206nm, sample size 10ul, PC appearance time is 6 ~ 7 minutes, detects purity 98% ~ 98.9%.
First the present invention extracts shrimp sauce with normal hexane from dry krill, through the acetone extraction removal of impurity, adopts silica gel column chromatography separating purification krill PC standard substance, detects, PC purity >=98% through high pressure liquid phase analysis.
The present invention has prepared standard of phosphatidylcholine in Euphausia superba, for contribution has been made in the detection of following krill deep processed product.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
Embodiment 1 prepares standard of phosphatidylcholine in Euphausia superba
Step is:
(1) dry krill 100g, adds normal hexane 600ml, stirs extraction 3 times, each 1h, often extracts once, filter and leached by dry krill at 20 DEG C; Merge the extracting solution (1700ml) of three times, at 30 DEG C, under-0.08Mpa, rotary evaporation removing normal hexane, obtains shrimp sauce 5.5g.
(2) in the shrimp sauce of above-mentioned preparation, add acetone 30ml, stir extraction at 25 DEG C 6 times, extract 0.5h at every turn, often extract once, filter to get filtrate and filter residue; Merge the filtrate (180ml) of 6 times, filtrate is at 30 DEG C, and rotary evaporation removing acetone under-0.08Mpa, must containing the oily sample liquid 4ml of phosphatide.
(3) take silica gel (200 ~ 300 order) 65g, 110 DEG C of activation 6h, add 195ml chloroform and stir into pulpous state in silica gel, adopt wet method dress post method that silica gel is encased in chromatography column (3 × 30cm
2) in, leave standstill, make silica gel fully be settled down to silica gel volume stability constant.
(4) get sample liquid 1.5ml prepared by step (2), slowly add in chromatography column, with chloroform: methyl alcohol=1:1(volume ratio) elution, flow velocity is 2.5ml/min, collects elutriant by every part of 100ml, concentrated.Thin-layer chromatography Qualitative Identification eluant component, merged by the concentrated solution with single PC component, evaporation of solvent, obtains PC standard substance 8mg, and detect through HPLC, PC purity is 98%.
The thin-layer chromatography qualitative checking method of Antarctic krill phosphatidyl choline (PC) is:
Thin-layer chromatography uses GF254 silica gel thin-layer plate, developping agent is chloroform: methyl alcohol: water: ethamine=14:6:0.6:5(V:V:V:V), exhibition, apart from being 6cm, gets the concentrated solution point sample of each elutriant, exhibition is dried after finishing, and puts into dibromothymolsulfonphthalein dye liquor cylinder dyeing 12 seconds; Dry after taking-up, dried thin layer plate is placed on thin layer chromatography scanner and observes: the Rf value of PC is 0.37.
The HPLC quantitative detecting method of Antarctic krill phosphatidyl choline (PC) is:
Adopt the U.S. (waters) HPLC instrument., 2489 UV-detector, SepaxHP-silica(4.6 × 250mm5um) and chromatographic column, make moving phase with methyl alcohol, flow velocity 1ml/min, column temperature 35 DEG C, determined wavelength 206nm, sample size 10ul, PC appearance time is 6 ~ 7 minutes, detects purity 98% ~ 98.9%.
Embodiment 2 prepares standard of phosphatidylcholine in Euphausia superba
Step is:
(1) dry krill 100g, adds normal hexane 500ml, stirs extraction 3 times, each 1h, often extracts once, filter and leached by dry krill at 25 DEG C; Merge the extracting solution (1350ml) of three times, at 45 DEG C, under-0.07Mpa, rotary evaporation removing normal hexane, obtains shrimp sauce 4.5g.
(2) in the shrimp sauce of preparation, add acetone 22.5ml, stir extraction at 20 DEG C 6 times, extract 0.5h at every turn, often extract once, filter to get filtrate and filter residue; Merge the filtrate (135ml) of 6 times, filtrate is at 40 DEG C, and rotary evaporation removing acetone under-0.07Mpa, must containing the oily sample liquid 4ml of phosphatide.
(3) take silica gel (200 ~ 300 order) 68g, 115 DEG C of activation 8h, add 204ml chloroform and stir into pulpous state in silica gel, adopt wet method dress post method that silica gel is encased in chromatography column (3 × 30cm
2) in, leave standstill, make silica gel fully be settled down to silica gel volume stability constant.
(4) get sample liquid 2ml prepared by step (2), slowly add in chromatography column, with chloroform: methyl alcohol=1:1.5 elution, flow velocity is 2ml/min, collects elutriant by every part of 300ml, concentrated, thin-layer chromatography Qualitative Identification eluant component.Merged by the concentrated solution with single PC component, evaporation of solvent, obtains PC standard substance 10mg, and detect through HPLC, PC purity is 98.5%.
Embodiment 3 prepares standard of phosphatidylcholine in Euphausia superba
Step is:
(1) dry krill 100g, adds normal hexane 550ml, stirs extraction 3 times, each 1h, often extracts once, filter and leached by dry krill at 35 DEG C; Merge the extracting solution (1500ml) of three times, at 35 DEG C, under-0.09Mpa, rotary evaporation removing normal hexane, obtains shrimp sauce 5g.
(2) in the shrimp sauce of preparation, add acetone 35ml, stir extraction at 35 DEG C 6 times, extract 0.5h at every turn, often extract once, filter and dry krill is leached; Merge the extracting solution (210ml) of three times, filtrate is at 40 DEG C, and rotary evaporation removing acetone under-0.09Mpa, must containing the oily sample liquid 4ml of phosphatide.
(3) take silica gel (200 ~ 300 order) 75g, 120 DEG C of activation 10h, add 225ml chloroform and stir into pulpous state in silica gel, adopt wet method dress post method that silica gel is encased in chromatography column (3 × 30cm
2) in, leave standstill, make silica gel fully be precipitated to volume stability constant.
(4) get sample liquid 3ml prepared by step (2), slowly add in chromatography column, with chloroform: methyl alcohol=1:2 elution.Flow velocity is 3ml/min every part 200ml, collects elutriant, concentrated, thin-layer chromatography Qualitative Identification eluant component.Merged by the concentrated solution with single PC component, evaporation of solvent, obtains PC standard substance 12mg.Detect through HPLC, PC purity is 98.9%.
Claims (2)
1. prepare a technique for standard of phosphatidylcholine in Euphausia superba, it is characterized in that: step is as follows:
(1) get dry krill 100g, add the normal hexane of 500 ~ 600ml, stir extraction at 20 ~ 35 DEG C 3 times, extract 1h at every turn, often extract once, filter and dry krill is leached; United extraction liquid, at 30 ~ 45 DEG C, rotary evaporation under-0.07 ~-0.09Mpa, obtains shrimp sauce;
(2) in the shrimp sauce of step (1) gained, add the acetone of 5 ~ 7 times, stir extraction at 20 ~ 35 DEG C 6 times, extract 0.5h, extracting liquid filtering at every turn, often extract once, filter to get filtrate and filter residue; Merge the filtrate of 6 times, at 30 ~ 40 DEG C, rotary evaporation under-0.07 ~-0.09Mpa, must containing the oily sample liquid of phosphatide;
(3) preparation of chromatography column: take 65 ~ 75g silica gel, activates 6 ~ 10h at 110 ~ 120 DEG C; In silica gel, add 3 times of chloroforms, stir into pulpous state; Adopt wet method dress post method that silica gel is encased in 3 × 30cm
2in chromatography column, leave standstill, make silica gel fully be settled down to volume stability constant;
(4) get step (2) gained containing the oily sample liquid 1.5 ~ 3ml of phosphatide, slowly join in chromatography column, with chloroform: methyl alcohol=1:1 ~ 2 elution, flow velocity is 2 ~ 3ml/min, collects elutriant by every part of 100 ~ 300ml;
(5) concentrated by each part elutriant collected, each part eluant component of thin-layer chromatography Qualitative Identification, merged by the concentrated solution of single for tool phosphatidyl choline component, evaporation of solvent, obtains phosphatidylcholine standard substance;
The thin-layer chromatography qualitative checking method of described Antarctic krill phosphatidyl choline is: thin-layer chromatography uses GF254 silica gel thin-layer plate, developping agent is chloroform: methyl alcohol: water: the volume ratio of ethamine is 14:6:0.6:5, exhibition distance is 6cm, get the concentrated solution point sample of each elutriant, exhibition is dried after finishing, and puts into dibromothymolsulfonphthalein dye liquor cylinder dyeing 12 seconds; Dry after taking-up, dried thin layer plate is placed on thin layer chromatography scanner and observes: the Rf value of phosphatidylcholine is 0.37;
Obtained phosphatidylcholine standard substance adopt HPLC method to carry out detection by quantitative, adopt watersHPLC instrument, 2489 UV-detector, SepaxHP-silica chromatographic column, makes moving phase with methyl alcohol, flow velocity 1ml/min, column temperature 35 DEG C, determined wavelength 206nm, sample size 10ul, PC appearance time is 6 ~ 7 minutes, detects purity 98% ~ 98.9%.
2. a kind of technique preparing standard of phosphatidylcholine in Euphausia superba according to claim 1, is characterized in that: described silica gel is 200 ~ 300 object silica gel.
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| CN106153763B (en) * | 2016-06-17 | 2018-12-14 | 浙江工商大学 | The hydrophilic chromatographic of phosphatide-tandem mass spectrum detection method in the new prawn of knife volume |
| CN108864179A (en) * | 2018-08-27 | 2018-11-23 | 大连工业大学 | A kind of egg PC preparation method based on low pressure column chromatography |
| CN109096327A (en) * | 2018-08-27 | 2018-12-28 | 大连工业大学 | A kind of preparation method of high-purity yolk phospholipid acyl ethanol amine |
| CN114965829A (en) * | 2022-04-28 | 2022-08-30 | 浙江工商大学 | Column chromatography method for enriching EPA/DHA type phosphatidylcholine |
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| CN101792461A (en) * | 2010-02-04 | 2010-08-04 | 南京工业大学 | Preparation process of soybean lecithin for injection |
| CN102041166A (en) * | 2011-01-20 | 2011-05-04 | 山东师范大学 | Method for extracting krill oil with high phosphatide content from Antarctic krills |
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