CN1223664C - Method of purifying high content DHA, EPA from fish oil - Google Patents

Method of purifying high content DHA, EPA from fish oil Download PDF

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CN1223664C
CN1223664C CN 03150169 CN03150169A CN1223664C CN 1223664 C CN1223664 C CN 1223664C CN 03150169 CN03150169 CN 03150169 CN 03150169 A CN03150169 A CN 03150169A CN 1223664 C CN1223664 C CN 1223664C
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dha
epa
fish oil
complex compound
content
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CN1478875A (en
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陶遵威
解红武
周运琴
刘惠芝
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TIANJIN INST OF MEDICAL AND MEDICINAL SCIENCES
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Abstract

The present invention discloses a method for obtaining high contents of DHA and EPA by separating and purifying ethylated products of fish oil with an AgNO3 solution method. With the method, products with the total content of DHA and EPA greater than 95% can be obtained, products with the content of DHA greater than 95% can be obtained, and products with the content of EPA higher than that of DHA can be obtained. The method of the present invention can reduce the cost of producing high contents of DHA and EPA of fish oil, can lower the toxic side effect of cash sales DHA and EPA products, and can improve the stability of DHA and EPA products.

Description

The method of purification high-content DHA, EPA from fish oil
Technical field
The present invention relates to a kind of method that obtains fat, oil or lipid acid from fat, oil or lipid acid therefrom through chemical modification.More particularly, the present invention relates to the method for purification high-content DHA, EPA from crude fish oil.
Background technology
People just find cardio-cerebral vascular disease patient a long time ago, the C in the erythrocyte 20-C 22Insatiable hunger lipid acid (Polyunsaturated fattiy acids PUFAS) content significantly be lower than the normal people, replenish C to patient 20-C 22PUFAS; patient's film fat is formed the generation noticeable change after several days; PUFAS significantly increases; blood fat descends thereupon; further affirmed PUFAS ratio and the significant regression relation of serum total cholesterol decline poling (r=0.9) in the meals to the mid-1960s Gunning etc.; low up to kayak cardiovascular morbidities in reported first Greenland such as 1978 Dyerberg with since ichthyophagy is relevant; eicosapentaenic acid in the fish oil (eicosapentaenoicacid) EPA; docosahexenoic acid (docosahexentaenoic acid) DHA just really causes people's attention; a large amount of studies confirm that; EPA; DHA has reducing blood-fat; platelet aggregation-against; relax thrombosis; effects such as the protection cerebrovascular; especially DHA can also increase brain function; improve differentiation power, memory and attention.DHA also can have an effect to the 26S Proteasome Structure and Function in neurocyte and the retina photoreception by the activity of annexin on brain and retina, this helps fetus, infant's brain pyramidal cell and the growth of rod cell, growth, maternity dress help the fetal brain cell development with DHA, particularly the premature infant takes DHA to safeguard the normal development of its rod photoreceptor cell, guarantees unlikely generation owing to lack sorry that eyesight that DHA causes weakens.Because human body self can not synthesize DHA, so development high purity DHA product meaning is very great.Because the effect of EPA and DHA is remarkable, the product that contains DHA, EPA goes on the market in a large number, but now sell product because content is not high, impurity is more, can draw superoxide poisoning, diarrhoea, maldigestion after taking, children taking can cause sexual prematurity, and the side effect of the big grade of taking dose especially is unfavorable for infant taking.
In order to improve EPA, DHA purity, Chinese scholars and manufacturer have carried out research widely.Main separation method has now: fatty enzyme process, urea adduct method, the crystallizing process under low temperature, metal salt precipitate method, supercritical extraction, vacuum fractionation method, molecular distillation method, silver nitrate solution extraction process and synthetic method etc.
For example people such as Yamguchi has reported and has adopted overcritical liquid phase chromatography separating high-purity DHA from fish oil, and purity can reach 95% to 99% (Yamguchi, Michihiro; Kadota, Yasuhiko; Tanaka, Isao; Et al.New separation method of high-purityDHA by supercritical fluid chromatography.Nihon Yukagakkai shi, 48 (10), 1169-1176 1999).People such as Liang have reported employing fat method from just separating EPA and DHA the fish viscera oil, and its purity can reach 58.3% (Liang.Jer-Hour; Hwang, Lucy Sun.Fractionation of squid Visceral oil ethyl ester by short-pathdistillation.J.Am.Oil Chem.Soc., 77 (7), 773-777,2000).People such as Gutierrez-Soto have reported the employing extraction process from fish oil purifying EPA and DHA, and its purity is greater than 72% (Gutierrez-Soto, Giselle; Herrera-Ramirez, Carlos H.Preparation ofconcentrates of eicosapentaenoic and docosahexenoic acids from fish oil.mg.Cienc.Quim.18 (2), 63-69 1998).People such as Haraldsson have reported that the fatty catalytic esterification method of employing prepares high-purity EPA and DHA from fish oil, and EPA and DHA total content can reach 50%, and DHA content is greater than 80%, and EPA is greater than 90% (Haraldsson, GudmundurG; Kristinsson, Bjorn; Sigurdarciottir.Ragnheidur; Et al.The preparationof concentrates of eicosapentaenoic acid and docosahexenoic acid bylipase-catalyzed transesterification of fish oil with ethanol.J.Am.OilChem.Soc., 74 (11), 1419-1424,1997).People such as Breivik have reported to adopt and have produced high-purity EPA and DHA from esterification process from fish oil that its content can reach 85% (Breivik.Harald; Haraldsson, Gudmundur G; Kristinsson.Bjorn.Preparation of highlypurified concentrates of eicosapentaenoic acid and docosahexenoic acid.J.Am.Oil Chem.Soc., 74 (11), 1425-1429,1997).People such as Nagahama have reported that the overcritical complexometry of employing Silver Nitrate separates EPA and DHA from fish oil, and the content of DHA can reach 90% (Nagahama, Kunio; Suzuki, Tatsuru; KiKuchi, Satoshi.et al.Separation of EPA and DHA from fish oil using supercritical extractionwith Ag complex pretreatment.Dev.Food Eng., Proc.mt.Congr.Eng.Food, 6th 1993.Pt.2,630-2 (Eng)).Rubin has reported that the employing extraction process separates EPA and DHA from natural resource, wherein mentions the employing column chromatography, uses AgNO 3And silica gel column chromatography, from Squid Oil, separate EPA and DHA, but do not see content report (Rubin.Methodof extraction and purifaction of polyunsaturated fatty acids fi-om naturalsources.US 4792418).People such as Nihonkagakushiryo have reported the DHA that adopts in the Silver Nitrate fractionating process separation purification fish oil, DHA content can reach more than 95%, this method can be used for suitability for industrialized production (Nihonkagakushiryo.Sophisticated purification of DHAethyl ester by silver nitrate fiactionation method for industrialization (DHA advanced purification/extraction technology research associationS) .DHA Kodo Seisci Chushutsu Gijutsu Kaihatsu Jigyo.Heisei 4-8Nendo.Kekka Gaiyo., 2002, PAGE.65-67).People such as Harima have reported preparation of industrialization and the separation method of high purity DHA, adopt purify DHA in the fish oil of silver nitrate solution extraction process, the content of DHA can reach (Harima Chem. more than 95%, Inc.Establishment of industrial manufacturing method for high-purity DHAester Examination of separation/collection of DHA ethyl ester andsolvents by silver nitrate method (DHA advanced purification extractiontechnology research association S) .DHA Kodo Seisei Chushutsu GijutsuKailiatsu Jio～vo.Heisci 4-8 Nendo.Kekka Gaiyo., 2002, PAGE.62-64).People such as Wu Guanghong are raw material with the mixing fish oil of yellow crucian carp after refining, adopt earlier through saltouing, use the synthetic method of inclusion again, extract EPA, DHA in the fish oil, purity reach 82% or more (Wu Guanghong, Liu Zhongfu. the research (3) of EPA, DHA in the synthetic method extraction fish oil: 28-29).Zhao Ya equality people has carried out Silver Nitrate complexing and supercritical CO 2The method that rectifying combines, the experimental study of from fish oil, extracting high purity EPA and DHA, its purity can reach 90% or more (Zhao Yaping, Wu Shouyi. the experimental study of extraction separation high purity EPA and DHA from fish is moored. engineering journal, 1997; 13 (4): 198-201).People such as Li He have reported the employing the crystallizing process under low temperature, enrichment EPA and DHA from fish oil fatty acid, through twice separation can reach 73%-79% (Lee and, Li Peiwen. the method 1997 of EPA and DHA in the low temperature crystallization enrichment fish oil; 16 (4): 50-52).People such as Wang Xuetong have reported employing RPLC technology, EPA in the fish oil and DHA have been carried out separation and purification, EPA that obtains and the content of DHA can rise and not reach (Wang Xuetong more than 98% and 86%, Zhang Shusheng. high performance liquid chromatography is separated EPA and the DHA. assay office in the preparation fish oil, 1999,18 (1): 42-45).People such as Zhu Shiyun have reported and have adopted urea adduct method that EPA in the fish oil of Dalian and DHA are carried out enrichment, EPA and DHA concentration and yield have reached (Zhu Shiyun more than 60% and 90% respectively in the enriched products, Bao Zonghong. EPA in the urea adduct method enrichment fish oil and the research of DHA. Chinese oil, 1997:22 (5): 54-56).People such as Hou Zhenrong have reported by low temperature-sylvite method and low temperature-acetone method enrichment EPA and DHA from anchovy, the total content of EPA and DHA reaches 71.56% (Hou Zhenrong, Song Xiujian, the research of preparation enrichment high purity unsaturated fatty acids from anchovy oil, China's pharmaceutical chemistry magazine, 1994,4 (4): 274-278).People such as Xue Changhu have reported that employing urea add on method is from extracting the unsaturated fatty acids (research of EPA and DHA from anchovy oil, do not see content report (Xue Changhu, Chen Xiubai. from anchovy oil, extract the research of unsaturated fatty acids (EPA and DHA). Chinese aquatic science, 1994,1 (1): 55-60).People such as Zhu Huixiang adopt urea adduct method to produce the patent report of high polyenoic acid ethyl ester content (EPA and DHA) refined fish oil technology from crude fish oil, EPA and DHA total content can reach 76.06% (Zhu Huixiang. from the technology that crude fish oil is produced the high polyenoic acid ethyl ester content refined fish oil, open day: 20000816.CN1263145).People such as Yang Qi have reported with the polyene fatty acid in the synthetic method extraction squid fish oil, promptly adopt the integrated application of methods such as salting-out process, low-temperature freezing, urea adduct method, EPA and DHA total content can reach (Yang Qi more than 70%, Yang Guane etc. (biological chemistry teaching and research room of Mountain Western Medicine S University). synthetic method is extracted the research of polyene fatty acid in the fish oil. Chinese marine drug, 2001,20 (5): 21-23).People such as Tao Hairong have reported with fish oil to be raw material, progressively purify through low temperature crystallization, underpressure distillation and tlc, obtain content respectively and be higher than 99% (methyl esters) EPA and DHA (methyl esters) (Tao Hairong, Lee and (department of chemistry of Beijing Normal University). the EPA of separating high-purity and DHA. China marine drug from fish oil, 2000,19 (5): 24-26).
But, more than method of the prior art or higher or all can not obtain highly purified DPA, DHA product easily because of complicated operation because of cost.For example, vacuum fractionation method separating effect is not ideal enough, the easy thermooxidizing excessively of product; The cost of molecular distillation equipment is higher, generally carries out the pyloric fat acid branch 20-30kg that per hour leaves school and calculates, and expense is about 1,300,000 yuan; The expense of supercritical extraction equipment is bigger; Urea adduct method is tighter to the experiment condition restriction, complex operation, generally under cold condition (30 ℃), the nitrate solution extraction process is difficult for carrying out mass production.
Summary of the invention
The present inventor at first works out the method for separating purifying EPA, DHA with Silver Nitrate water solvent method at home and abroad through crude fish oil extracting and purifying high content DHA, EPA are carried out big quantity research.
Method of the present invention is mainly finished by following steps: earlier with the crude fish oil ethyl esterization, pass through Ag again +The characteristic that forms complex compound with unsaturated double-bond makes DHA, EPA and the Ag that contains unsaturated double-bond in the fish oil +Reacting generating complex, and the mixture separation of DHA, EPA come out, Ag passed through then +(EPA) complex compound, Ag +(DHA) stability of complex compound is different, isolates the product that high-load DHA and EPA content are higher than DHA.
In the method for the invention, the raw material crude fish oil is the home-made crude fish oil, and can be selected from the crude fish oil of sardine oil, tuna oil, bonito oil, yellow crucian carp oil or anchovy oil.
In the method for the invention, can finish the esterification of crude fish oil according to the following steps: with crude fish oil, dehydrated alcohol, dense H 2SO 4Add reaction system, water-bath refluxes and reacts, and puts into separating funnel then, and standing over night is told the esterification layer, and reclaims ethanol.Wherein calculate, add dehydrated alcohol 800-2000ml, dense H with 1000 gram crude fish oils 2SO 4The 8-20 gram, the reaction times is 10 hours, obtains fish oil esterified prod 900-1125 gram.
In the method for the invention, can finish the preparation of Ag+ (EPA) complex compound, Ag+ (DHA) complex compound according to the following steps: with ethyl ester fish oil product, AgNO 3The aqueous solution adds reaction system, and room temperature reaction 3 hours is put into separating funnel, separates oil reservoir, gets Ag +(EPA) complex compound and Ag +(DHA) complex compound product reclaims unreacted ethyl ester fish oil.AgNO wherein 3The concentration of the aqueous solution is 50-80%, and calculates with 1000 gram ethyl ester fish oil, adds 500-1000 gram AgNO 3The aqueous solution.
In the method for the invention, can finish the preparation of EPA, DHA mixture according to the following steps: under the room temperature condition, toward Ag +(EPA) complex compound and Ag +(DHA) water of 10-20 times of volume of adding in the complex compound is used n-hexane extraction 3 times, and the organic layer anhydrous sodium sulfate drying reclaims the mixture that solvent obtains DHA-EPA.
In the method for the invention, can finish the separation preparation of high-content EPA according to the following steps: under the room temperature condition, with Ag +(EPA) complex compound and Ag +(DHA) complex compound is used n-hexane extraction 3 times, the organic layer anhydrous Na with 2-5 times of volume water dissolution 2SO 4Drying reclaims solvent.Wherein the content of EPA is 49% in the products obtained therefrom, and the content of DHA is 23.6%.
In the method for the invention, can finish the preparation of high-content DHA product according to the following steps: under the room temperature condition, with Ag +(EPA) complex compound and Ag +(DHA) complex compound dilute with water 2-5 doubly separates organic layer, and 20 times of water layer dilute with waters are used n-hexane extraction 3 times again.Wherein the content of EPA is 3.76% in the products obtained therefrom, and the content of DHA is 95.56%.
In the method for the invention, the Ag after can purifying with normal hexane or petroleum ether extraction +Residual solution is isolated organism, water layer in water-bath, be concentrated into the liquid level skinning cool crystallization, filter, AgNO 3Solid, the AgNO of recovery 3Solid can continue on for the separation of crude fish oil and purify.Wherein sherwood oil reclaims reusable.
Method of the present invention not only is adapted to the laboratory and separates the crude fish oil product, also can carry out large-scale separation of produced.Use method purifying EPA of the present invention, DHA not to need the investment of specific installation, technology is simple, cost is lower, the product purity height, and the reagent material of all uses is all recyclable, this method separation rate is different according to thick oil-contg difference, generally reaches 20% according to the crude fish oil total weight, EPA, DHA total content 90-100%.High-load DAH, EPA product can be eliminated and now sell the fish oil product because of the side effect that impurity caused, and make and now sell model change, and this is very great to improving the horizontal meaning of people ' s health.
Embodiment
Embodiment 1
1. material and instrument, physical and chemical index and detection method
1.1 material and reagent crude fish oil (Zhoushan fishery provides): dehydrated alcohol (AR Tianjin reagent two factories): Silver Nitrate (AR Tianjin reagent one factory): the vitriol oil (AR Tianjin reagent two factories): normal hexane (AR Beijing Chemical Plant)
1.2. laboratory apparatus
Separating funnel: electric mixer: magnetic stirring apparatus: round-bottomed bottle: triangular flask: rotating thin film evaporimeter: water-circulating pump: vacuum oil pump
1.3. products measure method DHA, EPA assay: vapor-phase chromatography
2. experimental section is a raw material with homemade crude fish oil, and first ethyl esterization is passed through Ag again +The characteristic that forms complex compound with unsaturated double-bond makes DHA, EPA and the Ag that contains unsaturated double-bond in the fish oil +Reacting generating complex, and the mixture separation of DHA, EPA come out, Ag passed through then +(EPA) complex compound, Ag +(DHA) stability of complex compound is different, isolates the product that high-load DHA and EPA content are higher than DHA.
2.1 the esterification of crude fish oil
With crude fish oil 1000g, dehydrated alcohol 800ml, dense H 2SO 48g adds reaction system, and water-bath refluxes, and reacts 10 hours, puts into separating funnel then, and standing over night is told the esterification layer, gets fish oil esterified prod 900g, wherein contains DHA 30.07%, and EPA 10.11%.Ethanol reclaims.
2.2 Ag +(EPA) complex compound, Ag +(DHA) preparation of complex compound
With ethyl ester fish oil product 40g, 70%AgNO 3Aqueous solution 40g adds reaction system, and room temperature reaction 3 hours is put into separating funnel, separates oil reservoir, gets Ag +(EPA) complex compound and Ag +(DHA) complex compound product reclaims unreacted ethyl ester fish oil.
2.3 the preparation of EPA, DHA mixture
Under the room temperature condition, will go up step gained Ag +(EPA) complex compound and Ag +(DHA) water of 20 times of volumes of adding in the complex compound, with normal hexane 50ml * 3 extractions, the organic layer anhydrous sodium sulfate drying reclaims the mixture 16g that solvent obtains DHA-EPA.Productive rate 20%.EPA 25.38%, and DHA 69.31%, total content 94.69%.
2.4 the separation of high-content EPA preparation
Under the room temperature condition, with homemade Ag +(EPA) complex compound and Ag +(DHA) complex compound is with 5 times of volume water dissolution, with normal hexane 20ml * 3 extractions, organic layer anhydrous Na 2SO 4Drying reclaims solvent, gets EPA (49%) DHA (23.6%) product 6g, total content 72.6%.
2.5 the preparation of high-content DHA product
Under the room temperature condition, with Ag +(EPA) complex compound and Ag +(DHA) the complex compound dilute with water is 5 times, separates organic layer, and 20 times of water layer dilute with waters are used normal hexane 50ml * 3 extractions again.DHA (95.56%) EPA (3.76%) product 10 grams, total content 99.32%.
2.6 Ag +Salt reclaims experiment
To contain Ag +Residual solution 300g isolates organism with petroleum ether extraction, (sherwood oil reclaim reusable) water layer in water-bath, be concentrated into the liquid level skinning cool crystallization, filter, AgNO 3Solid, AgNO 3The rate of recovery＞98%.The AgNO that reclaims 3Solid can continue on for the separation of crude fish oil and purify.
Embodiment 2
1. material and instrument, physical and chemical index and detection method
1.1 material and reagent crude fish oil (Zhoushan fishery provides): dehydrated alcohol (AR Tianjin reagent two factories): Silver Nitrate (AR Tianjin reagent one factory): the vitriol oil (AR Tianjin reagent two factories): normal hexane (AR Beijing Chemical Plant)
1.2. laboratory apparatus
Separating funnel: electric mixer: magnetic stirring apparatus: round-bottomed bottle: triangular flask: rotating thin film evaporimeter: water-circulating pump: vacuum oil pump
1.3. products measure method DHA, EPA assay: vapor-phase chromatography
2. experimental section is a raw material with homemade crude fish oil, and first ethyl esterization is passed through Ag again +The characteristic that forms complex compound with unsaturated double-bond makes DHA, EPA and the Ag that contains unsaturated double-bond in the fish oil +Reacting generating complex, and the mixture separation of DHA, EPA come out, Ag passed through then +(EPA) complex compound, Ag +(DHA) stability of complex compound is different, isolates the product that high-load DHA and EPA content are higher than DHA.
2.1 the esterification of crude fish oil
With crude fish oil 200g, dehydrated alcohol 400ml, dense H 2SO 44g adds reaction system, and water-bath refluxes, and reacts 10 hours, with 50ml * 5 washings, gets fish oil esterified prod 225g, wherein contains DHA 30.07%, and EPA 10.11%.
2.2 Ag +(EPA) complex compound, Ag +(DHA) preparation of complex compound
With ethyl ester fish oil product 80g, 70% AgNO 3Aqueous solution 40g adds reaction system, and room temperature reaction 3 hours is put into separating funnel, separates oil reservoir, gets Ag +(EPA) complex compound and Ag +(DHA) complex compound product reclaims unreacted ethyl ester fish oil.
2.3 the preparation of EPA, DHA mixture
Under the room temperature condition, will go up step gained Ag +(EPA) complex compound and Ag +(DHA) water of 10 times of volumes of adding in the complex compound, with normal hexane 50ml * 3 extractions, the organic layer anhydrous sodium sulfate drying reclaims the mixture 16g that solvent obtains DHA-EPA.Productive rate 20%.EPA 35.21％，DHA 58.22％。
2.4 the separation of high-content EPA preparation
Under the room temperature condition, with homemade Ag +(EPA) complex compound and Ag +(DHA) complex compound is with 2 times of volume water dissolution, with normal hexane 20ml * 3 extractions, organic layer anhydrous Na 2SO 4Drying reclaims solvent, gets EPA (49%) DHA (23.6%) product 5g.
2.5 the preparation of high-content DHA product
Under the room temperature condition, with Ag +(EPA) complex compound and Ag +(DHA) the complex compound dilute with water is 2 times, separates organic layer, and 20 times of water layer dilute with waters are used normal hexane 50ml * 3 extractions again.DHA (95.56%) EPA (3.76%) product 10 grams.
2.6 Ag +Salt reclaims experiment
To contain Ag +Residual solution 200g isolates organism with n-hexane extraction, (normal hexane reclaim reusable) water layer in water-bath, be concentrated into the liquid level skinning cool crystallization, filter, AgNO 3Solid, AgNO 3The rate of recovery＞98%.The AgNO that reclaims 3Solid can continue on for the separation of crude fish oil and purify.
3. cost efficient
We compare with commercially available 30%EPA, DHA U.S. Alaska fish oil with purifying EPA, DHA total content 90%: 60 yuan one bottle in commercially available U.S. Alaska fish oil (100 every 1g).The every kg 3-5 of homemade crude fish oil unit, 5 yuan of every kg of dehydrated alcohol (industry), 80 yuan of the every kg of normal hexane, the every 10g of Silver Nitrate, 100 yuan (reagent).The homemade crude fish oil of 1000g can be produced ethyl ester fish oil 1125g can get EPA, the product 225g of DHA total amount 90%.Because normal hexane, Silver Nitrate can reclaim substantially fully, ethanol recyclable 40%.So the product of every production 100g EPA, DHA total amount 90% needs the about 14.6-15.6 of cost unit, and 100g EPA, DHA total content equal 60 yuan of Alaska fish oil prices of 30%.As be converted to 30%EPA, the calculating of DHA total content, and the fish oil 100g of our development, cost is about 5 yuan.By contrast, we high-load EPA, the DHA of development necessarily have good competitive power on market, create huge economic benefit and social benefit.
Embodiment 3-5
AgNO with different concns 3Repeat the experiment of embodiment 1, experimental result sees the following form.
Embodiment 3 Embodiment 4 Embodiment 5
Extraction yield % color AgNO 3Concentration (%) DHA content (%) EPA content (%) total content (%) 17.6 yellowish 50 59.31 25.38 84.69 18.9 little yellow 60 62.08 27.92 90.00 20.1 colourless 80 69.31 25.38 94.69
Draw by above result, at ambient temperature AgNO 3-water law is separated the AgNO of DHA, EPA 3Optimum concn is 70%, and the purity height of extraction, product are colourless transparent solution.
We also confirm by pharmacological experiment, fish oil is under suitable dosage, not only can obviously improve the level of serum high-density LP cholesterol (HDL-G), can also obviously reduce the level of serum total cholesterol (TC), triglyceride level (TG) and low density lipoprotein cholesterol (LDL-C) simultaneously, show that fish oil has tangible reducing blood lipid to the experimental hyperlipidemia rat.The result confirms that also high-load fish oil and U.S.'s fish oil are suitable substantially to the effect for reducing fat of experimental hyperlipidemia rat.
Pharmacological experiment
Following pharmacological experiment study from homemade fish oil, isolate high-content DHA, EPA influence to the experimental hyperlipidemia rat fat, and tentatively compare with U.S. fish oil.
Laboratory animal: the Wistar rat, (X ± S), male and female half and half are provided by medical experiment animal development centre, Tianjin body weight 18.2 ± 6.0g, conformity certification number: No. the 006th, the moving word of doctor.
The experiment medicine: high-content fish oil of the present invention provides (content EPA 38.86%, DHA 54.42%) by synthetic chamber, Tianjin Institute of Medicine Science, and each component content of its 1g is suitable with U.S. fish oil 3g.
Alaska deep sea fish oil, U.S. T, T protective foods company produce (content EPA+DHA30%), and the U.S. is original-pack, the date manufactured: on June 16th, 1998.
Above fish oil compound method:
Take by weighing an amount of fish oil and be placed in the mortar, add a certain amount of tween 80 and grind, add water then and be mixed with emulsion and be for experiment, the administration volume is per kilogram of body weight 10ml.
Kang Li fat capsule is a Zhejiang Yue Zhou pharmaceutical Co. Ltd product, and lot number 990602 is mixed with suspension with distilled water, uses for experiment.
The total cholesterol test kit is produced by the Beijing Chemical Plant, lot number 990625.
Triglyceride determination test kit (enzymic colorimetric) is produced by the Beijing Chemical Plant, lot number: 990531.
The high density lipoprotein cholesterol test kit, Beijing Zhongsheng Biological Engineering High Technology Company produces, lot number: 990101.
The low density lipoprotein cholesterol test kit, Beijing Zhongsheng Biological Engineering High Technology Company produces, lot number: 990101.
Experimental technique: (70 of the rats of X ± S) are divided equally seven groups, and 10 every group, male and female are the normal control group for half and half, the first group, and feed and raise normal diet every day, irritates stomach simultaneously and give distilled water 10ml/kg.d to select body weight 18.2 ± 6.0g for use; Second group is high fat control group, and feed and raise high lipid food (yolk powder 10%, lard 5%, cholate 0.5%, normal diet 85%) every day, irritates stomach simultaneously and give distilled water 100ml/kg.d; Below five groups all feed and raise high lipid food, irritate stomach simultaneously and give different pharmaceutical, the 3rd group is high lipid food+fish oil 0.5g/kg.d; The 4th group is high lipid food+fish oil 1.0g/kg.d; The 5th group is high lipid food+fish oil 2.0g/kg.d; The 6th group is high lipid food+U.S. fish oil 3.0g/kg.d; The 7th group is high lipid food+Kang Li fat 300mg/kg.d (wherein fish oil 1.0g/kg group is suitable with DHA content with U.S. fish oil group EPA).Morning every day, gastric infusion in continuous 5 weeks, was weighed once weekly.After the last administration, all animal fasting 12 hours, then by the femoral artery bloodletting, centrifugal 20 minutes of 3000/min, separation of serum.With enzymatic assays total cholesterol (TC) content, triglyceride level (TG) content, adopt the precipitator method to measure high density lipoprotein cholesterol (HDL-C) content and low density lipoprotein cholesterol (LDL-C) content, compare between organizing then.
Experimental result:
TC, TG, HDL-C, LDL-C respectively organize measurement result and see Table 1.
Each experimental group body weight gain situation sees Table 2.
The influence of table 1 pair experimental hyperlipidemia rat fat (X ± S)
Group Number of animals (only) TC (mg/dl) TG (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl)
The high fat of the high fat control group of the Normal group+high fat of the fish oil 0.5g/kg+high fat of the fish oil 1.0g/kg+high fat of the fish oil 2.0g/kg+high fat of U.S. fish oil 3g/kg+Kang Li fat 0.3g/kg 10 10 10 10 10 10 10 88.13±9.16 186.93±27.09 △△△ 157.47±33.63 * 146.20±23.96 ** 138.67±20.87 *** 147.60±23.38 ** 146.67±21.33 ** 71.26±14.13 140.59±29.85 △△△ 106.67±3353 * 90.96±33.65 ** 87.26±20.5 *** 95 70±33.45 ** 91.56±29.05 ** 32.37±5.96 18.62±6.45 △△△ 21.81±5.12 25.90±8.12 * 28.82±8.00 ** 25.63±8.14 ** 28.48±5.66 ** 45.90±13.36 131.84±30.92 △△△ 107.51±32.55 95.18±29.41 * 84.91±23.59 ** 96.45±27.76 * 91.87±24.41 **
Annotate: 1. compare △ △ △ P＜0.001 with the normal control group
2. compare with high fat control group *P＜0.05, *P＜0.01, * *P＜0.001
Each experimental group body weight gain situation during table 2 administration (X ± S)
Group Number of animals (only) Body weight (g) before the administration Body weight after the administration (g) Rate of increase
1W 2W 3W 4W 5W
The normal control group 10 183.0±6.3 206.4±14.5 221.7±21.6 243.0±31.3 255.8±35.1 266.3±39.3 45.5
High fat control group 10 183.2±5.1 297.2±15.1 220.6±18.1 248.2±32.3 264.2±37.1 273.7±41.1 49.4
High fat+fish oil 0.5g/kg 10 180.4±6.5 194.4±17.8 210.6±13.8 229.2±22.4 244.4±28.2 260.2±35.7 44.2
High fat+fish oil 1.0g/kg 10 181.6±5.9 198.9±7.9 215.8±12.8 238.2±24.0 252.8±29.2 264.6±31.8 45.7
High fat+fish oil 2.0g/kg 10 182.6±6.3 198.0±7.7 215.4±12.1 236.0±21.0 252.6±29.7 264.6±34.1 44.9
High fat+U.S. fish oil 3g/kg 10 181.3±7.5 199.2±11.4 218.0±14.9 233.0±17.2 253.2±24.9 267.6±29.5 47.6
High fat+Kang Li fat 0.3g/kg 10 180.9±4.9 194.4±7.7 212.0±13.4 227.9±16.1 241.7±18.3 255.3±23.4 41.1
Table 1 as seen, high fat control group TC, TG, LDL-C are all apparently higher than the normal control group, HDL-C then is starkly lower than normal control group (P＜0.001).Positive control drug Kang Li fat group and high fat are compared, and the Serum HDL-C level is significantly improved, and serum TC, TG and LDL-C level then obviously reduce (P＜0.01).Above this experiment of presentation of results hyperlipidemia rats model is reliable.Fish oil 0.5,1.0,2.0g/kg group compare with high fat control group, all can obviously reduce TC, TG, and effect for reducing fat strengthen along with the raising of dosage.Table 1 is also shown in, fish oil 1.0,2.0kg/kg group and high fat control group relatively, can obviously improve HDL-C, reduce LDL-C (P＜0.05-0.001), when dosage is reduced to 0.5g/kg, not statistically significant.Table 1 confirms that also fish oil 1.0g/kg group is close substantially with U.S. fish oil 3.0g/kg group effect for reducing fat.
Table 2 as seen, each experimental group the weight of animals growth pattern no significant difference.
Conclusion:
1, fish oil has tangible reducing blood lipid to the experimental hyperlipidemia rat under suitable dosage.
2, high-content fish oil provided by the invention and U.S.'s fish oil are basic identical to the reducing blood lipid of experimental hyperlipidemia rat.
3, each experimental group the weight of animals growth pattern no significant difference.

Claims (5)

1, a kind of from crude fish oil the method for purification high-content DHA, EPA, it is characterized in that:, pass through Ag more earlier with the crude fish oil ethyl esterization +The characteristic that forms complex compound with unsaturated double-bond makes DHA, EPA and the Ag that contains unsaturated double-bond in the fish oil +Reacting generating complex, and the mixture separation of DHA, EPA come out, Ag passed through then +(EPA) complex compound, Ag +(DHA) stability of complex compound is different, isolates the product that high-load DHA and EPA content are higher than DHA; Wherein:
The esterification of crude fish oil may further comprise the steps: with crude fish oil, dehydrated alcohol, dense H 2SO 4Add reaction system, water-bath refluxes and reacts, and puts into separating funnel then, and standing over night is told the esterification layer, and reclaims ethanol;
Ag +(EPA) complex compound, Ag +(DHA) preparation of complex compound may further comprise the steps: with ethyl ester fish oil product, concentration is the AgNO of 50-80% 3The aqueous solution adds reaction system, and room temperature reaction 3 hours is put into separating funnel, separates oil reservoir, gets Ag +(EPA) complex compound and Ag +(DHA) complex compound product reclaims unreacted ethyl ester fish oil;
The preparation of EPA, DHA mixture may further comprise the steps: under the room temperature condition, toward Ag +(EPA) complex compound and Ag +(DHA) water of 10-20 times of volume of adding in the complex compound is used n-hexane extraction 3 times, and the organic layer anhydrous sodium sulfate drying reclaims the mixture that solvent obtains DHA-EPA;
The separation preparation of high-content EPA may further comprise the steps: under the room temperature condition, with Ag +(EPA) complex compound and Ag +(DHA) complex compound is used n-hexane extraction 3 times, the organic layer anhydrous Na with 2-5 times of volume water dissolution 2SO 4Drying reclaims solvent;
The preparation of high-content DHA product may further comprise the steps: under the room temperature condition, with Ag +(EPA) complex compound and Ag +(DHA) complex compound dilute with water 2-5 doubly separates organic layer, and 20 times of water layer dilute with waters are used n-hexane extraction 3 times again.
2, according to claim 1 a kind of from crude fish oil the method for purification high-content DHA, EPA, wherein in the esterif iotacation step of crude fish oil: calculate with 1000 gram crude fish oils, add dehydrated alcohol 800-2000ml, dense H 2SO 4The 8-20 gram, the reaction times is 10 hours, obtains fish oil esterified prod 900-1125 gram.
3, according to claim 1 and 2 a kind of from crude fish oil the method for purification high-content DHA, EPA, wherein Ag +(EPA) complex compound, Ag +(DHA) in the preparation process of complex compound: AgNO 3The concentration of the aqueous solution is 70%.
4, according to the method for claim 3 described a kind of purification high-content DHA, EPA from crude fish oil, Ag wherein +(EPA) complex compound, Ag +(DHA) in the preparation process of complex compound: calculate with 1000 gram ethyl ester fish oil, add 500-1000 gram AgNO 3The aqueous solution.
5, according to claim 1 and 2 a kind of from crude fish oil the method for purification high-content DHA, EPA, the Ag after wherein purifying with normal hexane or petroleum ether extraction +Residual solution is isolated organism, water layer in water-bath, be concentrated into the liquid level skinning cool crystallization, filter, AgNO 3Solid, the AgNO of recovery 3Solid can continue on for the separation of crude fish oil and purify.
CN 03150169 2003-07-21 2003-07-21 Method of purifying high content DHA, EPA from fish oil Expired - Fee Related CN1223664C (en)

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CN101049297B (en) * 2006-04-06 2010-11-24 北京百慧生化制药有限责任公司 High DNA type fatty ethyl ester in high purity, manufacturing method and preparation
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CN103962091B (en) * 2013-11-28 2017-01-25 大连工业大学 Method for separating EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) by using silver ion modified amino silica gel
CA3022744A1 (en) * 2016-05-02 2017-11-09 Nisshin Pharma Inc. Method for producing polyunsaturated fatty acid-containing composition
CN107162910B (en) * 2017-05-19 2019-12-31 陕西源邦生物技术有限公司 Method for preparing high-purity EPA-EE from fish oil
CN107629873B (en) * 2017-08-28 2020-08-25 浙江海洋大学 Method for enriching fish oil EPA and DHA through low-temperature crystallization
EP3586640A1 (en) 2018-06-21 2020-01-01 Nuseed Pty Ltd Dha enriched polyunsaturated fatty acid compositions
CN109912403A (en) * 2019-04-18 2019-06-21 南京市第一医院 A kind of method that Omega-3 prepares large conductance calcium activated potassium channel opener
CN115466180A (en) * 2022-09-13 2022-12-13 江苏海莱康生物科技有限公司 Method for purifying eicosapentaenoic acid ethyl ester

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