CN105985859A - Cod liver oil extract and preparation method thereof - Google Patents
Cod liver oil extract and preparation method thereof Download PDFInfo
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- CN105985859A CN105985859A CN201510059109.7A CN201510059109A CN105985859A CN 105985859 A CN105985859 A CN 105985859A CN 201510059109 A CN201510059109 A CN 201510059109A CN 105985859 A CN105985859 A CN 105985859A
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- 235000012716 cod liver oil Nutrition 0.000 title claims abstract description 66
- 239000003026 cod liver oil Substances 0.000 title claims abstract description 66
- 239000000284 extract Substances 0.000 title claims abstract description 44
- 238000002360 preparation method Methods 0.000 title claims abstract description 34
- 238000000605 extraction Methods 0.000 title claims abstract description 17
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 73
- 238000004821 distillation Methods 0.000 claims abstract description 22
- 238000004440 column chromatography Methods 0.000 claims abstract description 18
- 238000005554 pickling Methods 0.000 claims abstract description 14
- 238000007670 refining Methods 0.000 claims abstract description 13
- 239000002253 acid Substances 0.000 claims abstract description 7
- 238000000199 molecular distillation Methods 0.000 claims abstract description 6
- 239000000126 substance Substances 0.000 claims abstract description 5
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 105
- 210000004185 liver Anatomy 0.000 claims description 46
- 239000000243 solution Substances 0.000 claims description 34
- 241000251468 Actinopterygii Species 0.000 claims description 33
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 28
- 239000012141 concentrate Substances 0.000 claims description 27
- 239000003960 organic solvent Substances 0.000 claims description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 18
- 239000006228 supernatant Substances 0.000 claims description 18
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 14
- 238000007710 freezing Methods 0.000 claims description 14
- 230000008014 freezing Effects 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- 238000003756 stirring Methods 0.000 claims description 13
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 10
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 230000002378 acidificating effect Effects 0.000 claims description 9
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
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- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 9
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- 238000000926 separation method Methods 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical group [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
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- 239000003480 eluent Substances 0.000 claims description 6
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- 239000000463 material Substances 0.000 claims description 6
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 claims description 6
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 6
- 230000008719 thickening Effects 0.000 claims description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 6
- 238000000638 solvent extraction Methods 0.000 claims description 5
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- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
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- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 claims description 3
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- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 3
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- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 3
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- 235000011152 sodium sulphate Nutrition 0.000 claims description 3
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 3
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- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical compound CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 claims 1
- 229910052708 sodium Inorganic materials 0.000 claims 1
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- 239000007787 solid Substances 0.000 abstract description 5
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Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to a cod liver oil extract and a preparation method thereof. The preparation method comprises the steps of carrying out extraction, salting-out, acid pickling, distillation, acetone refining, molecular distillation, column chromatography, subzero fractionation and the like to remove impurities including proteins, fatty acids (free fatty acid and solid fatty acid), mineral matters, unsaponifiable matters, phospholipid, polypeptide, a low boiling-point solvent, cholesterol, polychlorinated biphenyl, carbides, big polar substances and the like, realize decolorization and improve the content of unsaturated fatty acid, thereby preparing a high-quality cod liver oil extract. The cod liver oil extract provided by the invention has reasonable components and proportions thereof, and the components achieve a synergetic effect, so that the effectiveness and safety of a cod liver oil preparation are significantly improved.
Description
Technical field
The invention belongs to field of medicine preparing technology, be specifically related to a kind of cod-liver oil extract and preparation method thereof.
Background technology
Cod-liver oil (COD LIVER OIL) is that the mankind grow the main source of necessary vitamin (VA, VD), be mainly used in preventing and treating vitamin A (VA) and vitamin D (VD) lack cause dry skin, follicular keratosis, scheroma, yctalopia, the illness such as rickets, with growing, highly important effect is had to human health.
The development of cod-liver oil can trace back to the sixties in 20th century, children rachitis disease rates in all parts of the country at that time is very high, the particularly incidence of disease of northern area is especially high, show as fontanel slowly do not close, the symptom such as cephalus quadratus, pigeon breast, bowlegs, these symptoms are not enough with calcium uptake, bone is soft relevant, and VD is for promoting the requisite active ingredient of calcium uptake.After rickets patient takes the fish liver oil preparations containing VA and VD, and get sun, play prevention and result for the treatment of well.
Cod-liver oil is the cod-liver oil extract preparing in albacore liver, by prepared cod-liver oil crude product through 0 DEG C of freezing, after sloughing fraction solids fat, obtain the higher cod-liver oil of concentration with refining of edible vegetable oil, it is mainly composed of VA and VD, possibly together with other vitamins, omega-fatty acid (such as DHA, EPA), AKGs ether, squalene isoreactivity composition.
At present, press " Chinese Pharmacopoeia " relevant regulations, commercially available medicinal cod-liver oil or vitamin AD are high concentration cod-liver oil or vitamin A and calciferol or D3, add refining of edible vegetable oil (sloughing hard fat at about 0 DEG C) or cod-liver oil dissolves and adjusts concentration, and add appropriate stabilizer and make, and the vitaminAD preparation (also referred to as fish oil type cod-liver oil) of the vitaminAD preparation of vegetable oil preparation and tradition fish oil preparation.
Vegetable oil matrix type drops is the substitute of cod-liver oil, cod-liver oil or Artificial Fish liver oil also known as Prof. Du Yucang, have an advantage in that: vegetable oil is abundant with the raw material resources of synthetic vitamin AD, steady sources, processing simplicity, but have that trophism is not enough, between composition without defects such as synergistic functions, possess hardly promotion intelligence development, collaborative strengthen immunity, collaborative improve retina, improving health etc. acts on.
Traditional fish oil extracting method includes cooking process, cooking process, enzymatic isolation method, supercritical extract, solvent extraction etc., and combines the ancillary technique raising extraction efficiencies such as ultrasonic wave, microwave, direct current, pulse.
1st, cooking process (fish oil Study on Extraction Method is in progress, " aquatic science and technology ", the third phase: 6-10 in 2009): homogenate, mixing, adds suitable quantity of water, heating a period of time at 40-60 DEG C, heat is centrifugal or filters, and obtains crude fish oil, with hot wash, centrifugal, it is dried.
2nd, light alkali hydrolysis method: homogenate, adds suitable quantity of water, adds appropriate alkaline liquor, heating, adds appropriate salt, centrifugal, prepares crude fish oil.
3rd, enzymatic isolation method: blend, heats under uniform temperature, adds protease or utilizes self enzyme, and enzymolysis is centrifugal, obtains crude fish oil.
4th, supercritical extract: fish liver crushes the extraction of thing ethyl acetate, crude extract processed, refines, obtain fish oil after concentration.
5th, (animal fat extracts and process technology progress solvent extraction, " fats and oils processing ", the 12nd phase of volume 35 in 2010,8-11 page；The detection of the extraction of fish oil and EPA and DHA in globefish livers, " Liaoning Journal of Traditional Chinese Medicine ", the 3rd phase of volume 36 in 2009,438-440 page): utilize petroleum ether, ether etc. to extract fish oil, do not add molecular clock and column chromatography technology.
There is the problems such as composition combination is unreasonable in tradition fish oil type fish liver oil preparations in active ingredient or nutritional labeling and its application, and there is the hidden danger such as security, validity, as infant crowd is excessively used EPA, can cause the possibility of intracorporeal organ bleeding.
How correctly to arrange in pairs or groups active component in cod-liver oil, the health demand to meet different groups especially pediatric population is particularly important.As worldwide resource constantly fails, albacore liver oil raw material becomes more and more valuable, how effectively to utilize cod-liver oil active ingredient, becomes technical problem in the urgent need to address.
Content of the invention
It is an object of the invention to provide the preparation method of a kind of cod-liver oil extract, comprise the steps:
1) extract: fish liver is made fish liver pulp, add and the immiscible organic solvent of water in fish liver pulp again, stirring, extraction, stands, and separates, take organic solvent layer, wherein, fish liver: the mass volume ratio of organic solvent is 1-2: 2-4, described organic solvent is selected from ethyl acetate, petroleum ether, n-hexane, any one or a combination thereof of ether；
2) saltout: in organic solvent extraction layer, add acid-base modifier, regulate pH8-9, stirring, stand, filter, take supernatant, prepare fish liver pulp and saltout supernatant；
3) pickling: after the solution washing of supernatant acidic materials of saltouing fish liver pulp, adds acid-base modifier in its supernatant, and regulation pH is 6-9, prepares fish liver pulp pickling supernatant；
4) distill: concentrate fish liver pulp pickling supernatant, prepare cod-liver oil distillation and concentration thing；
5) acetone refining: add acetone in cod-liver oil distillation and concentration thing, after mixing, cooling is placed, and filters, and filtrate concentrates, and prepares cod-liver oil acetone refining concentrate；
6) molecular clock: cod-liver oil extraction concentrate is carried out multiple-grade molecular distillation, prepares cod-liver oil molecular clock concentrate；
7) column chromatography: cod-liver oil molecular clock concentrate is dissolved in flowing phase, carry out separating wash-out with chromatographic column, collect and merge column chromatography eluent, concentrate, prepare cod-liver oil column chromatography concentrate, wherein, described flowing is selected from ethyl acetate mutually, acetone, isopropanol, ethanol, n-hexane, any one or a combination thereof of ether, described chromatographic column filler is selected from aluminum oxide, silica gel, macroreticular resin, any one or a combination thereof of ion exchange resin, described eluent is selected from ethyl acetate, acetone, isopropanol, ethanol, n-hexane, any one or a combination thereof of ether；
8) freezing separation: cod-liver oil column chromatography concentrate carries out under the conditions of≤10 DEG C freezing, places, and separates, and collects and separates liquid, to obtain final product.
In the preferred technical solution of the present invention, step 1) in fish liver: the mass volume ratio of organic solvent is 1: 1-2.
In the preferred technical solution of the present invention, step 1) described in organic solvent selected from any one or a combination thereof of ethyl acetate, acetone.
In the preferred technical solution of the present invention, step 1) described in extraction times be no less than 1 time, preferably not less than 2 times, be more preferably no less than 3 times.
In the preferred technical solution of the present invention, step 2) or step 3) described in acid-base modifier selected from sodium carbonate, sodium sulphate, NaOH, potassium hydroxide, potassium carbonate, potassium sulfate, sodium acetate, potassium acetate, sodium phosphate, any one or a combination thereof of potassium phosphate.
In the preferred technical solution of the present invention, step 3) described in acidic materials selected from hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid, any one or a combination thereof of citric acid.
In the preferred technical solution of the present invention, the concentration≤0.1N (equivalent) of described acidic aqueous solution.
In the preferred technical solution of the present invention, step 3) washing times of the middle acid substance aqueous solution is no less than 2 times, and preferably not less than 3 times.
In the preferred technical solution of the present invention, step 4) in thickening temperature be 30-100 DEG C, preferably 40-85 DEG C, more preferably 50-65 DEG C.
In the preferred technical solution of the present invention, step 4) described in concentration progression be no less than 2 grades, preferably not less than 3 grades.
In the preferred technical solution of the present invention, step 4) described in thickening temperature be not less than 40 DEG C, preferably not lower than 50 DEG C, be more preferably not less than 60 DEG C.
In the preferred technical solution of the present invention, step 5) described in the distillation progression of multiple-grade molecular distillation be no less than 2 grades, preferably not less than 3 grades.
The preferred technical solution of the present invention, step 5) in first order molecular clock temperature be 80 DEG C-170 DEG C.
The preferred technical solution of the present invention, step 5) in second level molecular clock temperature be 90 DEG C-195 DEG C.
The preferred technical solution of the present invention, step 5) in third level molecular clock temperature be 100 DEG C-205 DEG C.
In the preferred technical solution of the present invention, step 8) described in cryogenic temperature≤5 DEG C, be preferably≤0 DEG C.
Another object of the present invention is to provide a kind of cod-liver oil extract, the preparation method of described cod-liver oil extract comprises the steps:
1) extract: fish liver is made fish liver pulp, add and the immiscible organic solvent of water in fish liver pulp again, stirring, extraction, stands, and separates, take organic solvent layer, wherein, fish liver: the mass volume ratio of organic solvent is 1-2: 2-4, described organic solvent is selected from ethyl acetate, petroleum ether, n-hexane, any one or a combination thereof of ether；
2) saltout: in organic solvent extraction layer, add acid-base modifier, regulate pH8-9, stirring, stand, filter, take supernatant, prepare fish liver pulp and saltout supernatant；
3) pickling: after the solution washing of supernatant acidic materials of saltouing fish liver pulp, adds acid-base modifier in its supernatant, and regulation pH is 6-9, prepares fish liver pulp pickling supernatant；
4) distill: concentrate fish liver pulp pickling supernatant, prepare cod-liver oil distillation and concentration thing；
5) acetone refining: add acetone in cod-liver oil distillation and concentration thing, after mixing, cooling is placed, and filters, and filtrate concentrates, and prepares cod-liver oil acetone refining concentrate；
6) molecular clock: cod-liver oil extraction concentrate is carried out multiple-grade molecular distillation, prepares cod-liver oil molecular clock concentrate；
7) column chromatography: cod-liver oil molecular clock concentrate is dissolved in flowing phase, carry out separating wash-out with chromatographic column, collect and merge column chromatography eluent, concentrate, prepare cod-liver oil column chromatography concentrate, wherein, described flowing is selected from ethyl acetate mutually, acetone, isopropanol, ethanol, n-hexane, any one or a combination thereof of ether, described chromatographic column filler is selected from aluminum oxide, silica gel, macroreticular resin, any one or a combination thereof of ion exchange resin, described eluent is selected from ethyl acetate, acetone, isopropanol, ethanol, n-hexane, any one or a combination thereof of ether；
8) freezing separation: cod-liver oil column chromatography concentrate carries out under the conditions of≤10 DEG C freezing, places, and separates, and collects and separates liquid, to obtain final product.
In the preferred technical solution of the present invention, step 1) in fish liver: the mass volume ratio of organic solvent is 1: 1-2.
In the preferred technical solution of the present invention, step 1) described in organic solvent selected from any one or a combination thereof of ethyl acetate, acetone.
In the preferred technical solution of the present invention, step 1) described in extraction times be no less than 1 time, preferably not less than 2 times, be more preferably no less than 3 times.
In the preferred technical solution of the present invention, step 2) or step 3) described in acid-base modifier selected from sodium carbonate, sodium sulphate, NaOH, potassium hydroxide, potassium carbonate, potassium sulfate, sodium acetate, potassium acetate, sodium phosphate, any one or a combination thereof of potassium phosphate.
In the preferred technical solution of the present invention, step 3) described in acidic materials selected from hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid, any one or a combination thereof of citric acid.
In the preferred technical solution of the present invention, the concentration≤0.1N (equivalent) of described acidic aqueous solution.
In the preferred technical solution of the present invention, step 3) washing times of the middle acid substance aqueous solution is no less than 2 times, and preferably not less than 3 times.
In the preferred technical solution of the present invention, step 4) in thickening temperature be 30-100 DEG C, preferably 40-85 DEG C, more preferably 50-65 DEG C.
In the preferred technical solution of the present invention, step 4) described in concentration progression be no less than 2 grades, preferably not less than 3 grades.
In the preferred technical solution of the present invention, step 4) described in thickening temperature be not less than 40 DEG C, preferably not lower than 50 DEG C, be more preferably not less than 60 DEG C.
In the preferred technical solution of the present invention, step 5) described in the distillation progression of multiple-grade molecular distillation be no less than 2 grades, preferably not less than 3 grades.
The preferred technical solution of the present invention, step 5) in first order molecular clock temperature be 80 DEG C-170 DEG C.
The preferred technical solution of the present invention, step 5) in second level molecular clock temperature be 90 DEG C-195 DEG C.
The preferred technical solution of the present invention, step 5) in third level molecular clock temperature be 100 DEG C-205 DEG C.
In the preferred technical solution of the present invention, step 8) described in cryogenic temperature≤5 DEG C, be preferably≤0 DEG C.
Another object of the present invention is to provide cod-liver oil extract for preparing the application in the nutritional agents of replenishing vitamins A and D or medicine.
In order to clearly state protection scope of the present invention, following term is defined by the present invention:
Molecular clock of the present invention refers to a kind of distillating method operating under a high vacuum, and the mean free path of vapour molecule, more than the distance between evaporating surface and condensing surface, utilizes the difference of each component evaporation rate in feed liquid to carry out separation of liquid mixtures.
ω 3 unrighted acid of the present invention refers in polyunsaturated fatty acid that first unsaturated bond occurs in the 3rd of carbochain methyl end.
Unless otherwise stated, when the present invention relates to the percentage between liquid and liquid, described percentage is volume/volume percentage；When the present invention relates to the percentage between liquid and solid, described percentage is volume/weight percentage；When the present invention relates to the percentage between solid and liquid, described percentage is weight/volume percent；Remaining is weight/percentage by weight.
Compared with prior art, the present invention has a following Advantageous Effects:
1st, the present invention by extracting, saltouing, pickling, distillation, acetone refining, molecular clock, column chromatography, the step such as freezing separation, remove the impurity such as the protein in fish oil extract, aliphatic acid (free fatty, solid fatty acid), mineral matter, unsaponifiable matter, phosphatide, polypeptide, low boiling point solvent, cholesterol, Polychlorinated biphenyls, carbide, big polar substances, decolour and improve the content of unrighted acid, thus preparing high-quality cod-liver oil extract.Composition in the cod-liver oil extract of the present invention and reasonable mixture ratio thereof, have synergistic function between composition.
2nd, compared with traditional fish liver oil preparations, the cod-liver oil extract of the present invention has intelligence development, enhancing immunity, beneficially fetus, infantile health growth and the brain cell development promoting children, promote the effects such as visual development, significantly increase validity and the security of fish liver oil preparations.
Brief description
The finger-print of Fig. 1 tuna liver oil.
Detailed description of the invention
Illustrating the present invention below with reference to embodiment, embodiments of the invention are merely to illustrate technical scheme, and the essence of the non-limiting present invention.
Embodiment 1The preparation of tuna cod-liver oil extract
1) extract: weigh the tuna fish liver that 200kg rubs, add 400kg ethyl acetate, stirring, extract 80 minutes, take ethyl acetate layer；Again extract 80 minutes with 240kg ethyl acetate, take ethyl acetate layer；Merge, prepare ethyl acetate extraction phase 625L；
2) saltout: in ethyl acetate extraction phase, add the saturated Na of 125L2CO3, adjust PH to 8, stir 5 minutes, stand 0.5h；
3) pickling: after washing three times with 0.1NHCL, according to 1kg Na2SO4/ 50L ethyl acetate adds Na2SO4, overnight；
4) distill: under the conditions of 40-50 DEG C, distillation and concentration 10h, obtain 40L fish oil；Under the conditions of 85 DEG C, rotary evaporation distills 12h, prepares 5.3L fish oil crude product；
5) acetone refining: add 10L acetone in fish oil crude product, mix, under the conditions of 0-5 DEG C, after standing 2.5h, filters, filtrate distillation and concentration, obtains 4.7L fish oil；
6) molecular clock: by step 5) fish oil for preparing carries out secondary molecules distillation, and wherein, first order molecular distills, and low temperature is 80 DEG C, and high temperature is 170 DEG C, distills 8h；Secondary molecules distills, and low temperature is 80 DEG C, and high temperature is 195 DEG C, distills 9h, obtains 4.3L fish oil；
7) column chromatography: by step 6) prepare fish oil be dissolved in 4.3L ethyl acetate after, after carrying out glass chromatography column process, take ethyl acetate chromatography thing, distillation and concentration, prepare cod-liver oil 4L.
8) freezing separation: by step 7) fish oil for preparing is positioned at 5 DEG C freezing 5 hours, at 5 DEG C, uses plate and frame filter press press filtration, prepare cod-liver oil 3.9L.
Embodiment 2The preparation of dogfish oil extract
1) extract: weigh the shark hepatic that 100kg rubs, add 250kg ethyl acetate, stirring, extract 60 minutes, take ethyl acetate layer；Add 140kg ethyl acetate, stirring, extract 60 minutes, take ethyl acetate layer, merge, prepare ethyl acetate extract 320L；
2) saltout: in ethyl acetate extract, add the saturated Na of 70L2CO3, adjust PH to 9, stir 10 minutes, stand 1h；
3) pickling: after washing three times with 0.2NHCL, according to 1.5kg Na2SO4/ 50L ethyl acetate adds Na2SO4, overnight；
4) distilling: under the conditions of 40-50 DEG C, distillation and concentration 10h, obtaining 25L fish oil solution, under the conditions of 85 DEG C, rotary evaporation distills 12h, prepares 3L fish oil crude product；
5) acetone refining: add 8L acetone in 3L fish oil crude product, mix, after standing 3h at 0-4 DEG C, filters, filtrate rotary evaporation, prepares 2.5L fish oil；
6) molecular clock: by step 5) fish oil for preparing carries out secondary molecules distillation, and wherein, first order molecular distills, and low temperature is 80 DEG C, and high temperature is 170 DEG C, distills 8h；Secondary molecules distills, and low temperature is 80 DEG C, and high temperature is 195 DEG C, distills 9h, obtains 2.3L fish oil；
7) column chromatography: by step 6) prepare fish oil be dissolved in 3L ethyl acetate after, after carrying out glass chromatography column process, take ethyl acetate chromatography thing, distillation and concentration, prepare cod-liver oil 2L.
8) freezing separation: by step 7) fish oil for preparing is positioned at 10 DEG C freezing 5 hours, at 10 DEG C, filters with supercentrifuge, remove filter residue, prepare cod-liver oil 3.8L.
Embodiment 3The preparation of mackerel cod-liver oil extract
1) extract: weigh the mackerel fish liver that 200kg rubs, add 400kg ethyl acetate, stirring, extract 100 minutes, take ethyl acetate layer；Extract 100 minutes with 250kg ethyl acetate again, take ethyl acetate layer；Merge, prepare ethyl acetate layer 650L；
2) saltout: in ethyl acetate layer, add the saturated Na of 130L2CO3, adjust PH to 8, stir 10 minutes, stand 0.5h；
3) pickling: after washing three times with 0.1NHCL, according to 1kg Na2SO4/ 50L ethyl acetate adds Na2SO4, overnight；
4) distill: under the conditions of 50-60 DEG C, distillation and concentration 5h, obtain 38L fish oil；Under the conditions of 95 DEG C, rotary evaporation distills 6h, prepares 6L fish oil crude product；
5) acetone refining: add 12L acetone in prepared fish oil crude product, mix, after standing 1h at 0-4 DEG C, filters, filtrate rotary evaporation, prepares 5L fish oil；
6) molecular clock: by step 5) fish oil for preparing carries out secondary molecules distillation, and wherein, first order molecular distills, and low temperature is 80 DEG C, and high temperature is 170 DEG C, distills 8h；Secondary molecules distills, and low temperature is 80 DEG C, and high temperature is 195 DEG C, distills 9h, obtains 4.5L fish oil；
7) column chromatography: by step 6) prepare fish oil be dissolved in 3L ethyl acetate after, after carrying out glass chromatography column process, take ethyl acetate chromatography thing, distillation and concentration, prepare cod-liver oil 4.2L.
8) freezing separation: by step 7) fish oil for preparing is positioned at 0 DEG C freezing 5 hours, at 0 DEG C, filters, prepare cod-liver oil 3.9L.
Embodiment 4The quality testing of cod-liver oil extract of the present invention
Detection method:
Chromatographic column: 5 μm of 250 × 4.6mm of Diamonsil C18 post
Flowing phase: acetonitrile: isopropanol=6: 4
Flow velocity: 1.0ml/min
Detection wavelength: 258nm
After testing, the natural complex (VA, VD) that is mainly composed of in the cod-liver oil extract (Fig. 1 is shown in by its finger-print) of tuna of the present invention forms, wherein, VA is mainly made up of VA-DHA ester (structure is shown in formula I), VA-EPA ester (structure is as shown in Formula II), VA-DHA ester: the mass ratio of VA-EPA ester is 6: 4；VD is mainly made up of VD-DHA ester (structure is as shown in formula III), VD-EPA ester (structure is as shown in formula IV), wherein VD-DHA ester: the mass ratio of VD-EPA ester is 8-9: 2-1.
Embodiment 5The influence research to immune function of mice for the tuna liver oil extract of the present invention
30 ICR mouse, are randomly divided into three groups, often organize 10.Giving self-control tuna liver oil 20ml/kg body weight (high dose group), 10ml/kg body weight (low dose group) respectively, control group gives common commercially available tuna liver oil (20ml/kg body weight).Every day gavage once, continuous 7 days, recorded mice serum hemolysin content, spleen and thymic weight in the 7th day.
The impact on immune function of mice for the table 1 tuna of the present invention liver oil extract
* P ＜ 0.05, * * P ＜ 0.01, compares with commercially available tuna liver oil control group.
From table 1, compared with common commercially available tuna liver oil, rich in bioactivators such as VA-DHA, VA-EPA, VD-DHA, VD-EPA in the tuna liver oil extract of the present invention, have and build up health, foster growth, brain tonic, intelligence development, improve immunologic function, promote mouse immune organ weight to increase and serum hemolysin increase etc. acts on, and has raising immunity function.
Embodiment 6The influence research to mouse visual development for the dogfish oil extract of the present invention
40 female mices, at candlelight with 2: 1 ratios, and 20 male mices, with cage mating, are found cloudy bolt person secondary morning and are designated as pregnant 0 day.
Pregnant mouse is randomly divided into four groups, often organizes 10.Giving dogfish oil 2.5ml/kg body weight (high dose group), 0.5ml/kg body weight (middle dosage group), 0.1ml/kg body weight (low dose group) (being equivalent to DHA1000mg/kg body weight, 200mg/kg body weight, 40mg/kg body weight) respectively, control group gives commercial plant Oleovitamin A and D preparation (2.5ml/kg body weight).Every day gavage once.Often group dams was from pregnant 0 day, raised with synthetic diet.Within pregnant 5 days, rise, within every two days, adjust gavage amount by body weight accordingly.After childbirth, dams gavage amount increases to 1.5 times of pregnancy period.
Record newborn mouse widens the view age in days, visual development is tested and is carried out in 16 ages in days, catch newborn mouse afterbody, upside down, it is allowed to face with foreleg towards the edge of a plane, be gradually shortened distance, if having caught edge with fore paw before antenna touches edge, prove that visual development is normal, otherwise be abnormal, record each group of newborn mouse visual development normal number.
The impact on newborn mouse visual development for the table 2 dogfish oil of the present invention extract
* P ＜ 0.05, compares with vegetable oil vitaminAD preparations. Control group.
With commercial vegetable oil vitaminAD formulations Comparative, the age in days of widening the view of the dosage group newborn mouse high, middle of the dogfish oil of the present invention is early than low dose group and control group, and difference has conspicuousness (P ＜ 0.05)；The normal percentage of high, medium and low dosage group 16 age in days visual development is higher than control group, and difference also has significant (P ＜ 0.05).Illustrate that the dogfish oil extract of the present invention can remarkably promote newborn mouse visual development.
Claims (15)
1. a preparation method for cod-liver oil extract, comprises the steps:
1) extract: fish liver is made fish liver pulp, then adds and the immiscible organic solvent of water in fish liver pulp,
Stirring, extraction, stand, separate, take organic solvent layer, wherein, fish liver: the quality volume of organic solvent
Ratio is for 1-2: 2-4, described organic solvent selected from ethyl acetate, petroleum ether, n-hexane, ether any one
Or a combination thereof；
2) saltout: in organic solvent extraction layer, add acid-base modifier, regulate pH8-9, stirring, stand,
Filter, take supernatant, prepare fish liver pulp and saltout supernatant；
3) pickling: after the solution washing of supernatant acidic materials that fish liver pulp is saltoutd, in its supernatant
Adding acid-base modifier, regulation pH is 6-9, prepares fish liver pulp pickling supernatant；
4) distill: concentrate fish liver pulp pickling supernatant, prepare cod-liver oil distillation and concentration thing；
5) acetone refining: add acetone in cod-liver oil distillation and concentration thing, after mixing, cooling is placed,
Filtering, filtrate concentrates, and prepares cod-liver oil acetone refining concentrate；
6) molecular clock: cod-liver oil extraction concentrate is carried out multiple-grade molecular distillation, prepares cod-liver oil molecule and steam
Evaporate concentrate；
7) column chromatography: cod-liver oil molecular clock concentrate is dissolved in flowing phase, carries out separating wash-out with chromatographic column,
Collect and merge column chromatography eluent, concentrate, prepare cod-liver oil column chromatography concentrate, wherein, described flowing
Mutually selected from ethyl acetate, acetone, isopropanol, ethanol, n-hexane, any one or a combination thereof, institute of ether
The chromatographic column filler stated is selected from aluminum oxide, silica gel, macroreticular resin, any one or its group of ion exchange resin
Close, described eluent selected from ethyl acetate, acetone, isopropanol, ethanol, n-hexane, ether arbitrary
Plant or a combination thereof；
8) freezing separation: cod-liver oil column chromatography concentrate carries out under the conditions of≤10 DEG C freezing, places, point
From collection separates liquid, to obtain final product.
2. preparation method according to claim 1, step 1) in fish liver: the quality volume of organic solvent
Ratio is 1: 1-2.
3. preparation method according to claim 1 and 2, step 1) described in organic solvent be selected from second
Acetoacetic ester, any one or a combination thereof of acetone.
4. the preparation method according to any one of claim 1-3, step 1) described in extraction times not
It less than 1 time, preferably not less than 2 times, is more preferably no less than 3 times.
5. the preparation method according to any one of claim 1-4, step 2) or step 3) described in
Acid-base modifier is selected from sodium carbonate, sodium sulphate, NaOH, potassium hydroxide, potassium carbonate, potassium sulfate, vinegar
Acid sodium, potassium acetate, sodium phosphate, any one or a combination thereof of potassium phosphate.
6. the preparation method according to any one of claim 1-5, step 3) described in acidic materials choosing
From hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid, any one or a combination thereof of citric acid.
7. the preparation method according to any one of claim 1-6, the concentration≤0.1N of described acidic aqueous solution.
8. the preparation method according to any one of claim 1-7, step 3) the middle acid substance aqueous solution
Washing times be no less than 2 times, preferably not less than 3 times.
9. the preparation method according to any one of claim 1-8, step 4) in thickening temperature be
30-100 DEG C, preferably 40-85 DEG C, more preferably 50-65 DEG C.
10. the preparation method according to any one of claim 1-9, step 4) described in thickening temperature
It is not less than 40 DEG C, preferably not lower than 50 DEG C, more desirably not less than 60 DEG C.
11. preparation methods according to any one of claim 1-10, step 5) described in many-level molecule
The distillation progression of distillation is no less than 2 grades, preferably not less than 3 grades.
12. preparation methods according to any one of claim 1-11, step 5) in first order molecular clock
Temperature is 80 DEG C-170 DEG C, and second stage preferably molecular clock temperature is 90 DEG C-195 DEG C, and the more preferably third level is divided
Sub-vapo(u)rizing temperature is 100 DEG C-205 DEG C.
13. preparation methods according to any one of claim 1-12, step 8) described in cryogenic temperature
It≤5 DEG C, is preferably≤0 DEG C.
14. 1 kinds of cod-liver oil extracts, are prepared by the method described in any one of claim 1-13.
15. are prepared cod-liver oil extract by the method described in any one of claim 1-13 supplements dimension for preparing
Application in the nutritional agents of raw element A and D or medicine.
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