CN106117044A - The method separating the alltrans fatty acid with anti-tumor activity from antarctic krill oil - Google Patents
The method separating the alltrans fatty acid with anti-tumor activity from antarctic krill oil Download PDFInfo
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Abstract
The invention provides a kind of method separating the alltrans fatty acid with anti-tumor activity from antarctic krill oil.Antarctic krill oil silica gel column chromatography coupling preparative high-performance liquid chromatographic is carried out isolated and purified, adjunct antineoplastic Activity determination determines effective ingredient, determining the molecular weight of effective ingredient, structure composition and configuration respectively through high-resolution electrospray ionization mass spectrometry, nuclear magnetic resonance, NMR and laser raman scattering spectrum, identifying effective ingredient is alltrans EPA and alltrans DHA.The median lethal concentration of kinds of tumor cells is found by relatively Antarctic krill, commercially available bathypelagic fish oil, Amur Acipenser Sinensis liver EPA and DHA, the alltrans EPA and alltrans DHA in antarctic krill oil source has the effect of excellent antitumor cell growth, and to normal control cells without obvious inhibiting effect, there is the DEVELOPMENT PROSPECT of potential antitumor drug candidate.
Description
Technical field
The present invention relates to a kind of method separating the natural E type fatty acid with anti-tumor activity from antarctic krill oil,
Identified alltrans EPA and DHA is to human breast carcinoma, hepatocarcinoma, early children's grain leukemia, chronic myelocytic leukemia, tissue
Cell lymphoma etc. have the effect of significant antitumor cell growth.
Background technology
In in the past few decades, the sickness rate of tumor is in ascendant trend year by year, even if there being the continuous progressive of medical skill,
Tumor remains a big cause of disease of human death's peacetime.Research worker studies generation and the development of tumor from different directions,
And develop the three big therapies based on actinotherapy (radiotherapy), chemotherapy (chemotherapy) and surgical operation therapy (operation),
But all there is drawback in various degree in three big therapies.There is significant side effect in radiotherapy, such as inappetence, alopecia, drowsiness, the thinnest
Born of the same parents reduce, and some patients is also with the situation such as hemorrhage；Operative therapy is preferable to tumorigenic early stage or prometaphase effect, but swollen
After tumor spreads, the effect of operative therapy is just deteriorated, and operative therapy is the biggest to psychology and the physiological load of patient, has
A little patients can cause sequela or complication because of operation, and operative therapy can only be for solid tumor, to non-physical tumor such as blood
Cancers etc. are helpless.The medicine of chemotherapy is the most, gradually forms synthetic drug, Chinese medicine or the series such as natural product, bio-pharmaceutical and produces
Product, but many antitumor drug exist the drawbacks such as poor specificity, after some medicine enters in the patient, killing tumor cell same
Time kill normal cell in the patient the most in a large number, cause series of side effects, such as alopecia, vomit, feel sick and inappetence etc.,
Therefore develop the medicine that a class safety is high, side effect is little, antitumous effect is good be people in the urgent need to, the most unsaturated
The research and development of fatty acid once got more and more people's extensive concerning.
Fatty acid is fat, phospholipid and the main component of glycolipid, is the material base of life.Whether contain according in its carbochain
Unsaturated bond or double bond is had to be divided into satisfied fatty acid and unsaturated fatty acid, the fatty acid quilt containing two or more unsaturated bond
It is referred to as polyunsaturated fatty acid, as eicosapentaenoic acid (EPA) and docosahexenoic acid (DHA) contain five and six respectively
Double bond.There is again cis (Z-type) and trans (E type) not according to its configuration fatty acid, common EPA and DHA in bathypelagic fish oil
It is Z-type it is considered to be ω-3 race fatty acid of great nutritive value and physiologically active.There are some researches show, Z-EPA and Z-
DHA has enhancing human immunity, antiallergic and promotes the important function such as intracellular fatty acid metabolism, and DHA is to infant god
Also significant role is played through phylogeny.Since nineteen seventies, research worker finds that Z-EPA and Z-DHA has
The ability of suppression kinds of tumor cells growth, such as the median lethal concentration to people early children grain acute leukemia cells system HL60
(IC50) it is respectively 85 μMs and 70 μMs, the IC to human breast carcinoma cell lines MCF-750It is respectively 85 ± 5 μMs and 65 ± 5 μMs；They
Main Function mechanism be inducing apoptosis of tumour cell or cause growth of tumour cell Cycle Arrest.Present stage due to Z-EPA and
Z-DHA the most just must can produce lethal effect to kinds of tumor cells, causes its application to be very limited.
Trans (E type) fatty acid naturally occurs and manually manufactures two types, as contained 4-in the milk products such as milk from cows and goats
The E type fatty acid or ester of 9%, hydrogenated oil and fat is then a kind of unsaturated fatty acid that vegetable oil carries out hydrogenation modification generation, contains
There is the E type fatty acid of 14.2-34.3%；There is an E type carbon-carbon double bond and be also categorized as E type fat in conjugated linoleic acid (CLA)
Acid.There is bigger dispute in the biological function of this kind of fatty acid, especially at two aspects seeming opposition carcinogenic and for cancer.Grind
Study carefully and show that CLA can also have research to think that it can accelerate the pernicious process of breast carcinoma with adjuvant treatment of breast cancer, separately have research to think
Non-correlation between the two, fails to form final conclusion so far.To the research of E type fatty acid mainly based on E-oleic acid, research is thought
The high intake of E-oleic acid can increase the risk of cardiovascular disease, especially coronary heart disease, but to early growth and development, II type glycosuria
The diseases such as disease, hypertension, cancer and the dependency of trans fats, be able to verify that currently without positive evidence.E type EPA and DHA make
For the isomer of Z-type EPA and DHA, the most not yet have been reported that.
Antarctic krill oils and fats rich in polyunsaturated fat, especially omega-3 unsaturated fatty acid, wherein EPA and DHA content
Up to more than 20%.It is generally believed that EPA and DHA in Antarctic krill is Z-type, there is antioxidation, defying age, antitumor
Deng effect, but actually it mostly is and analogizes, lack experimental basis.The present invention uses solvent extraction, silica gel column chromatography, prepares efficient liquid phase
Antarctic krill oils and fats has been carried out isolated and purified by the methods such as chromatograph, by high-resolution electrospray ionization mass spectrometry (HRESIMS), nuclear-magnetism
Two single components of technical appraisement such as resonance, Raman spectrum are E type EPA and DHA, and it is main city that its anti-tumor activity is better than Z-type
Selling fish oil EPA and DHA, the exploitation for new type antineoplastic medicine provides new clue.
Summary of the invention
It is an object of the invention to provide a class and there is good antitumor action, and that safety is high, side effect is little is anti-swollen
Tumor agent alltrans polyunsaturated fatty acid, E type EPA and DHA.With mixed extractant solvent, silica gel column chromatography, preparative liquid chromatography
Isolated and purified Antarctic krill oils and fats has the active component of anti-tumor activity, by gas chromatography-mass spectrography, liquid phase color
The structure of the Instrumental Analysis effective ingredient such as spectrum-mass spectrometry, high-resolution electrospray ionization mass spectrometry, nuclear magnetic resonance, NMR, Raman spectrum, and
Investigate they impacts on kinds of tumor cells growth, and compare with commercially available fish oil EPA and DHA and Acipenser Sinensis liver EPA and DHA
Relatively.
The present invention provides a kind of alltrans EPA or alltrans DHA with anti-tumor activity, and it divides from antarctic krill oil
From obtaining.Common EPA and DHA in bathypelagic fish oil is Z-type, E type EPA and DHA as the isomer of Z-type EPA and DHA,
Anti-tumor activity is the most not yet had to report.
The present invention also provides for separating natural alltrans EPA or the alltrans with anti-tumor activity from antarctic krill oil
The method of DHA, it comprises the steps:
(1) by antarctic krill oil addition to silicagel column carries out column chromatography for separation, collect eluent, remove solvent, obtain
Fraction I and fraction II, fraction I is yellow oil, and fraction II is cerise grease；
(2) fraction II dilution in acetonitrile, separates by high performance preparative liquid chromatography method, with acetonitrile and the mixed solvent of water
System carries out eluting mutually for flowing, collects eluting gained different component, removes solvent, obtains composition--1 successively to II-5, all
For the colourless or oily mater of yellowish, wherein composition--2 is alltrans EPA, and composition--4 is alltrans DHA.
Further, in the column chromatography for separation described in step (1), described eluent be volume ratio be 7:3-9:1
Normal hexane and ether mixed liquor, adding percent by volume in mixed liquor is the acetic acid of 1-5%.
Further, the particle diameter of the silica gel described in step (1) is 300～400 mesh, silica gel and the quality of antarctic krill oil
Ratio is 30～100:1, preferably 50～80:1, more preferably 60～70:1, and chromatographic column ratio of height to diameter is 10～16:1.
Further, the chromatographic column filler described in step (2) is anti-phase C18, particle diameter 10 μm, the elution program of flowing phase
As follows: between eluting 0-10min, with 62-68% acetonitrile eluting；Between eluting 10-20min, wash with 67-73% acetonitrile
De-；Between eluting 20-60min, with 72-78% acetonitrile eluting, the elution program of preferred flowing phase is as follows: eluting 0-
Between 10min, with 65% acetonitrile eluting；Between eluting 10-20min, with 70% acetonitrile eluting；Between eluting 20-60min, use
75% acetonitrile eluting.
In above-mentioned separation method, antarctic krill oil loading is to silicagel column, and uses eluent to carry out eluting, collects eluting
Liquid, the volume of every fraction is the 1/20～1/40 of chromatographic column retention volume, preferably 1/30, detect by thin layer chromatography method simultaneously
The material of opposed polarity contained in gained eluent, merges the eluent after testing with identical polar fraction, in temperature 20-
40 DEG C, pressure is rotary evaporation removal solvent under conditions of-0.08～-0.1Mpa, respectively obtains fraction I and fraction II, fraction I
For yellow oil, fraction II is cerise grease.Anti-tumor activity testing result display fraction II has stronger resisting and swells
Tumor activity, uses the further separate fraction of preparative high-performance liquid chromatographic post II.
In above-mentioned separation method, the fraction II of silica gel column chromatography isolated uses reverse C further18Efficiently prepare liquid phase
Chromatography separates, and is flowing phase with the mixed solvent of acetonitrile and water, and the flow velocity of flowing phase is not particularly limited, ability
Field technique personnel can select suitable flow rate of mobile phase, it may be preferred to 10～20mL/min, more according to this area routine techniques
Preferably 15mL/min.Light absorption value at ultraviolet detection 210nm, carries out Fractional Collections according to appearance time, in temperature 20-40
DEG C, pressure is rotation additional issue recycling design under conditions of-0.08～-0.1Mpa, obtains composition--1 successively to composition--5.Anti-
Tumor promotion testing result display composition--2 and II-4 has stronger anti-tumor activity.Composition--2 is adopted with composition--4
With HRESIMS,1H-NMR、13C-NMR, laser raman scattering spectrum etc. carry out Structural Identification, determine that composition--2 is for alltrans
EPA, composition--4 is alltrans DHA.
In the present invention, described antarctic krill oil is prepared as follows obtaining: Antarctic krill freezes shrimp or euphausia superba powder
For raw material, it is stirred at room temperature 2h, by mutually rotated for organic solvent evaporation with mixed solvent (normal hexane: ethanol=10:1, v/v)
Euphausia superba sauce is i.e. can get after concentration.In order to obtain more free EPA and DHA, enzymatic isolation method hydrolysis South Pole phosphorus can be used
Shrimp sauce fat, concrete enzymatic hydrolysis condition is: 1.0g antarctic krill oil, 1.0g deionized water and 20mg immobilized-lipase (Novozym
435) being placed in 15mL centrifuge tube, then react 24 hours under the conditions of lucifuge in 30 DEG C of shaking tables, reaction uses chlorine after terminating
Imitative extracting enzymolysis solution, then carry out isolated and purified.Antarctic krill oil can also use commercially available product.
The present invention also provides for alltrans EPA described above or alltrans DHA application in preparing antitumor drug.Excellent
Selection of land, described tumor is human breast carcinoma, hepatocarcinoma, early children's grain leukemia, chronic myelocytic leukemia, histiocytic pouring
Bar tumor or carcinoma of prostate.
The present invention also provides for including above-mentioned alltrans EPA and pharmaceutically acceptable adjuvant, or includes above-mentioned alltrans DHA
Pharmaceutical composition with pharmaceutically acceptable adjuvant.Preferably, described pharmaceutical composition is granule, tablet or capsule.
The method have the advantages that
The present invention uses method isolated from antarctic krill oil of silica gel column chromatography coupling preparative liquid chromatography to have anti-
The alltrans EPA (E-EPA) or alltrans DHA (E-DHA) of tumor promotion.Common EPA and DHA in bathypelagic fish oil is Z-type
, E type EPA and DHA, as the isomer of Z-type EPA and DHA, the most not yet have been reported that.E-EPA and E-DHA pair of Antarctic krill
Kinds of tumor cells has the killing ability of excellence, and its effect is all better than commercially available fish oil and EPA and DHA in sturgeon liver oil source,
And to normal liver cell HL7702 without obvious growth inhibited, there are the potentiality of exploitation new type antineoplastic medicine.
Accompanying drawing explanation
Fig. 1 is the thin-layer chromatogram representing opposed polarity fraction；
Fig. 2 is the preparative high-performance liquid chromatographic figure representing silica gel column chromatography fraction II；
Fig. 3 is the growth inhibited curve representing and preparing-2 couples of MCF-7 of composition-；
Fig. 4 is the growth inhibited curve representing and preparing-4 couples of MCF-7 of composition-；
Fig. 5 is the mass spectrum representing composition--2 (EPA)；
Fig. 6 is the mass spectrum representing composition--4 (DHA)；
Fig. 7 represents composition--2 (EPA)1H-NMR schemes；
Fig. 8 represents composition--4 (DHA)1H-NMR schemes；
Fig. 9 represents composition--2 (EPA)13C-NMR schemes；
Figure 10 represents composition--4 (DHA)13C-NMR schemes；
Figure 11 is the laser raman scattering spectrogram representing Antarctic krill with commercially available fish oil EPA；
Figure 12 is the laser raman scattering spectrogram representing Antarctic krill with commercially available fish oil DHA.
Detailed description of the invention
Following non-limiting example can make those of ordinary skill in the art that the present invention be more fully understood, but not with
Any mode limits the present invention.In following embodiment, if no special instructions, the experimental technique used is conventional method, institute
All can buy from biological or chemical company with material, reagent etc..
The detailed description of the invention of the present invention relates to following experimental technique and analysis method:
1. cell is cultivated
Human breast carcinoma cell lines MCF-7 and MDA-MB-231, Bel7402 SMMC-7721, people early children's grain is acute in vain
Before disorders of blood cell line HL60, human chronic polymorpho nuclear leukemia cells system K562, human tissue cell's property lymphoma cell line U937, people
Row gland cell system PC-3 and people normal liver tissue cell HL7702 is purchased from Chinese Academy of Sciences's cell bank, wherein MCF-7 and
The used culture medium of MDA-MB-231 is DMEM high glucose medium (Hyclone), and the used culture medium of remaining cell is RPMI-
1640 culture medium (Hyclone), all add 10%FBS (Hyclone) and 1% Pen .-Strep mixed liquor in incubation
(Hyclone).All cells is all at 25cm2Culture bottle is cultivated.Above-mentioned cell is cultivated two days later in culture bottle, attached cell
Respective handling is carried out respectively: attached cell adds a certain amount of trypsin solution, so after PBS cleans with suspension cell
The corresponding culture medium of rear addition is blown and beaten the most gently and is disperseed to cell, uses cell counting count board to calculate cell number；Suspension cell warp
Remove supernatant after 800r/min centrifugation, use PBS to wash twice again resuspended by corresponding culture medium, then use thin
Born of the same parents' counting chamber counts.Above-mentioned cell is added 96 orifice plates, and the cell number controlling each hole is 3000, the culture volume in each hole
It is 200 μ L, 96 orifice plates is put into cell culture incubator and hatches 24 hours.
2. anti-tumor activity detection
The impact on different growth of tumour cell of the isolated and purified material is detected, to determine with mtt assay or CCK-8 test kit
Its anti-tumor activity.The detection of attached cell uses mtt assay, and suspension cell uses CCK-8 test kit.Test substance is first dissolved
Detect again after DMSO, using the culture medium containing 0.5%DMSO as comparison.First will add determinand according to suitable concentration
96 orifice plates of matter or comparison liquid are placed in cell culture incubator and hatch 16-18 hour, add 20 μ L MTT or CCK-8 (Kai Ji) to each hole
Reagent, then under 570nm (MTT)/450nm (CCK-8) and 630nm, detect light absorption value respectively after hatching 4 hours.Cell survival rate
Calculate as follows:
Cell survival rate (mtt assay, %)=[experimental group light absorption value (A570-A630) meansigma methods/matched group light absorption value (A570-
A630) meansigma methods] * 100
Cell survival rate (CCK-8 method, %)=[experimental group light absorption value (A450-A630) meansigma methods/matched group light absorption value
(A450-A630) meansigma methods] * 100
Median lethal concentration IC50Calculating use improvement karber's method formula:
lg(IC50)=Xm-I*(P-(3-Pm-Pn)/4)
Wherein XmFor lg (maximum drug level), I is lg (maximum drug level/second highest drug level), and P is that experimental group is thin
Born of the same parents' survival rate sum, PmFor maximum cell survival rate, PnFor the smallest cell survival rate.
3. the extraction of antarctic krill oil:
First shrimp or euphausia superba powder are frozen as raw material with Antarctic krill, with mixed solvent (normal hexane: ethanol=10:1, v/
V) it is stirred at room temperature 2h, extracts shrimp sauce in raw material, i.e. can get Antarctic krill by after mutually rotated for organic solvent evaporation and concentration
Shrimp sauce.Similarly, from Acipenser Sinensis fish liver, oils and fats is extracted with mixed solvent.
The separation of various components in embodiment 1 antarctic krill oil
(1) preparation of silicagel column: it is 3.0cm that the 300-400 mesh silica gel wet method after being activated by 300g loads internal diameter, a length of
In the glass detached dowel of 35.0cm, balance with normal hexane.
(2) take euphausia superba sauce 5g, add and carry out column chromatography for separation to ready silicagel column, use eluent successively
A (normal hexane: ether: acetic acid=80:20:1.5, v/v/v) and eluent B (chloroform: methanol: water=60:30:5, v/v/v) enters
Row eluting, every fraction is 10mL, the opposed polarity of contained substance in thin layer chromatography detection eluent, as it is shown in figure 1, in figure with
Antarctic krill oil is as comparison, and numeral i, ii and iii corresponding triglyceride or polarity connection respectively are bordering on the material of triglyceride,
Oleic acid or polarity connection are bordering on the material of oleic acid, phospholipid substance.Merge according to thin layer chromatography result and there is evaporating of identical polar
Point, finally there are 4 groups of fractions, wherein fraction I affords through eluent A with fraction II, and fraction III with fraction IV is
Affording through eluent B, the cumulative volume collecting four kinds of fractions is respectively 510,420,285 and 225mL, by each fraction warp
Rotary evaporation concentrates standby, obtain after vacuum rotary steam concentrates the quality of four kinds of fractions be respectively 2.78,1.12,0.59 and
0.17g.Wherein, fraction I is yellow oily material, and fraction II is cerise oily mater, and fraction III is dark red with fraction IV
Color oily mater.The thin layer chromatography condition of Fig. 1 is: use silica gel thin-layer chromatography plate, light with pencil at 1cm bottom chromatoplate
Standardized bar point line-transect, is placed in collected each reception equidistant point sample of pipe eluent in the chromatography cylinder containing developing solvent on a line-transect
Chromatographing, developing solvent is eluent A or eluent B, and Fig. 1 (a), 1 (b) and 1 (c) used developing solvent are eluent A, 1 (d)
Used developing solvent is eluent B, can reach preferably Detection results under these conditions.Through the antitumor to tumor cell
Activity determination, fraction II is to people's early children grain acute leukemia cells system HL60, human tissue cell's property lymphoma cell line U937 tool
There is growth inhibited effect, and to Human normal hepatocyte HL7702 without growth inhibited effect, other three kinds of fractions are all to above-mentioned cell
Without obvious effect.
(3) purification of fraction II
Fraction II 1:3 dilution in acetonitrile, the mixing by volume that will obtain in step (2), then be splined on and prepare high-efficient liquid
In phase chromatographic column, wherein chromatographic column is that YMC prepares post (YMC-Pack ODS-A, 20 × 250mm, particle diameter 10 μm), each sample introduction
0.6mL, with acetonitrile-water system as eluent, with the speed isorheic elution of 15mL/min, elution program is: 0-10 minute, uses
65% acetonitrile solution eluting；10-20 minute, with 70% acetonitrile solution eluting；20-60 minute, with 75% acetonitrile eluting.Purple
Light absorption value at outer detection 210nm, its spectrogram is as shown in Figure 2.Carry out Fractional Collections according to appearance time, there are 5 kinds of materials,
Successively it is numbered II-1 to II-5 according to appearance time, removes solvent with Rotary Evaporators standby.It is diluted to suitable concn with DMSO
The detection various materials growth inhibited effect to MCF-7 of rear use mtt assay, testing result shows that II-2 deposits with II-4 couple of MCF-7
In growth inhibited effect (as shown in Figure 3,4) in various degree, and II-1, II-3 and II-5 couple of MCF-7 is without growth inhibited effect.
Fig. 3 result shows, the growth inhibited effect of-2 couples of MCF-7 of composition- improves along with the increase of concentration, at concentration 12.5 μ g/mL
Time suppression ratio reach 59%.The growth inhibited effect of Fig. 4 result display-4 couples of MCF-7 of composition- carries along with the increase of concentration
Height, when concentration 12.5 μ g/mL, suppression ratio reaches 71%.
(4) composition--2 and the Structural Identification of II-4
By the composition--2 obtained in step (3) and II-4 through HRESIMS,1H-NMR and13C-NMR detects, result
Such as Fig. 5-10.The ms fragment relative molecular mass of Fig. 5-6 explanation both materials is respectively 320.2570 and 346.2707,
Supposition may be for adding NH4+Peak, being computed the actual relative molecular mass of both materials is 302.2246 and 328.2402, respectively
It coincide with the relative molecular mass data of EPA and DHA；Two kinds of materials1H-NMR spectrum (Fig. 7-8) and EPA and DHA in document
's1H-NMR spectrum is more identical, and concrete outcome is shown in Table 1；Two kinds of materials13C-NMR spectrogram (Fig. 9-10) also reports number with document
According to unanimously, therefore can determine that composition--2 and II-4 is EPA and DHA in omega-3 polyunsaturated fatty acids respectively.
1. two kinds of active components of table II-2 and II-41The measurement result of H-NMR spectrum
Laser raman scattering spectrum (Figure 11, the 12) display of EPA Yu DHA in antarctic krill oil and commercially available fish oil, two kinds are not
The Raman spectrum of EPA and DHA in same source is at displacement 1658cm-1、1671cm-1、1265cm-1And 1303cm-1Exist everywhere substantially
Difference, at front two, displacement represents Z-type carbon carbon unsaturated double-bond and E type unsaturated double-bond respectively, then ratio [the ν of displacement at two
(1303cm-1)/ν(1265cm-1)] the biggest, represent that in sample, Z-type carbon-carbon double bond number is the most.Antarctic krill oil extracts
The raman scattering spectrum of EPA and DHA can't see obvious E type unsaturated double-bond, therefore EPA and DHA in antarctic krill oil
Z-type unsaturated carbon carbon double bond number be less than raman scattering spectrum detection limit, it can thus be assumed that this EPA and DHA is alltrans
, there are two kinds of unsaturated carbon-carbon double bonds of E Yu Z in EPA and DHA, Z-type will be far more than E type the most simultaneously in commercially available fish oil EPA and DHA.
The anti-tumor activity detection of embodiment 2 antarctic krill oil EPA Yu DHA
In antarctic krill oil, E type EPA of isolated and DHA have inhibitory action to the growth of kinds of tumor cells, its
IC50Value is shown in Table 2, and these numerical value are below matched group standard substance EPA and DHA, Acipenser Sinensis liver and EPA and DHA in commercially available fish oil source
Corresponding IC50Value, and compared with currently marketed many antitumor drug, Antarctic krill EPA Yu DHA is to Human normal hepatocyte
HL7702 is without overt toxicity, and therefore in application, it has bigger advantage, it may have wide application prospect.
Claims (8)
1. there is natural alltrans EPA or the separation method of alltrans DHA of anti-tumor activity, comprise the steps:
(1) by antarctic krill oil addition to silicagel column carries out column chromatography for separation, collect eluent, remove solvent, obtain fraction I
With fraction II, fraction I is yellow oil, and fraction II is cerise grease；
(2) fraction II dilution in acetonitrile, separates by high performance preparative liquid chromatography method, with acetonitrile and the mixed solvent system of water
Carry out eluting mutually for flowing, collect eluting gained different component, remove solvent, obtain composition--1 successively to II-5, wherein group
Dividing II-2 is alltrans EPA, and composition--4 is alltrans DHA.
Separation method the most according to claim 1, it is characterised in that in the column chromatography for separation described in step (1), described
Eluent be volume ratio be normal hexane and the ether mixed liquor of 7:3-9:1, in mixed liquor add percent by volume be 1-5%'s
Acetic acid.
Separation method the most according to claim 1, it is characterised in that the particle diameter of the silica gel described in step (1) be 300～
400 mesh, silica gel is 30～100:1 with the mass ratio of antarctic krill oil, and chromatographic column ratio of height to diameter is 10～16:1.
Separation method the most according to claim 1, it is characterised in that the high performance preparative liquid chromatography described in step (2)
Column packing is anti-phase C18, particle diameter 10 μm, the elution program of flowing phase is as follows: between eluting 0-10min, use 62-68% second
Nitrile eluting；Between eluting 10-20min, with 67-73% acetonitrile eluting；Between eluting 20-60min, with 72-78% acetonitrile eluting.
5. separation method as claimed in claim 1 obtains natural alltrans EPA or alltrans DHA are preparing antitumor drug
In application.
Application the most according to claim 6, it is characterised in that described tumor is that human breast carcinoma, hepatocarcinoma, early children's grain are white
Disorders of blood, chronic myelocytic leukemia, histiocytic lymphoma or carcinoma of prostate.
7. a pharmaceutical composition, it includes natural alltrans EPA and the pharmacy that separation method as claimed in claim 1 obtains
Upper acceptable adjuvant.
8. a pharmaceutical composition, it includes natural alltrans DHA and the pharmacy that separation method as claimed in claim 1 obtains
Upper acceptable adjuvant.
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| CN111362790A (en) * | 2020-04-17 | 2020-07-03 | 辽宁科技大学 | Chromatographic method for separating EPA and DHA |
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| CN105055402A (en) * | 2015-07-20 | 2015-11-18 | 西安膳方医药科技有限公司 | Composition of omega-3 series of poyenoic fatty acids and natural products |
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| CN105055402A (en) * | 2015-07-20 | 2015-11-18 | 西安膳方医药科技有限公司 | Composition of omega-3 series of poyenoic fatty acids and natural products |
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|---|
| KJETIL BERGE ET AL.: "Safety assessment of SuperbaTM krill powder:Subchronic toxicity study in rats", 《TOXICOLOGY REPORTS》 * |
| NIYAZI ACAR ET AL.: "FA Composition of Plasma, Red Blood Cell, and Liver Phospholipids of Newborn Piglets Fed trans Isomers of α-Linolenic Acid", 《LIPIDS》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111362790A (en) * | 2020-04-17 | 2020-07-03 | 辽宁科技大学 | Chromatographic method for separating EPA and DHA |
| CN111362790B (en) * | 2020-04-17 | 2022-12-13 | 辽宁科技大学 | Chromatographic method for separating EPA and DHA |
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