CN106117044B - Method for isolating all-trans fatty acids with antitumor activity from Antarctic krill oil - Google Patents

Abstract

The invention provides a method for isolating all-trans fatty acid with antitumor activity from Antarctic krill oil. The Antarctic krill oil was separated and purified by silica gel chromatography coupling to prepare high performance liquid chromatography, and the anti-tumor activity was assisted to determine the active ingredients. The active ingredients were determined by high-resolution electrospray ionization mass spectrometry, nuclear magnetic resonance and laser Raman scattering spectra. According to the molecular weight, structural composition and configuration, the active ingredients were identified as all-trans EPA and all-trans DHA. Comparing the median lethal concentrations of Antarctic krill, commercially available deep-sea fish oil, and Amur sturgeon liver EPA and DHA on various tumor cells, it was found that all-trans EPA and all-trans DHA derived from Antarctic krill oil have excellent anti-tumor cell growth and has no obvious inhibitory effect on normal control cells, and has potential development prospects for anti-tumor drug candidates.

Description

Method for isolating all-trans fatty acids with antitumor activity from Antarctic krill oil

technical field

The invention relates to a method for isolating natural E-type fatty acids with anti-tumor activity from Antarctic krill oil. The identified all-trans EPA and DHA are effective against human breast cancer, hepatocellular carcinoma, promyelocytic leukemia, and chronic myeloid cell carcinoma. Leukemia, histiocytic lymphoma, etc. have significant anti-tumor cell growth effects.

Background technique

In the past few decades, the incidence of tumors has been increasing year by year. Even with the continuous advancement of medical technology, tumors are still a major cause of human death in peacetime. Researchers study the occurrence and development of tumors from different directions, and have developed three major therapies, mainly radiation therapy (radiotherapy), chemotherapy (chemotherapy) and surgical therapy (surgery), but there are varying degrees of differences in the three major therapies. disadvantages. Radiation therapy has significant side effects, such as loss of appetite, hair loss, lethargy, leukopenia, and some patients also have bleeding. Surgical therapy has a better effect on the early or mid-stage of tumor occurrence, but after the tumor has spread, the effect of surgical therapy The stool becomes worse, and the psychological and physical burden of surgical therapy on patients is relatively large, and some patients may cause sequelae or complications due to surgery, and surgical therapy can only be used for solid tumors, and it is powerless for non-solid tumors such as blood cancers. There are many drugs for chemotherapy, and a series of products such as synthetic drugs, traditional Chinese medicines or natural products, and biological drugs have gradually formed. However, many anti-tumor drugs have disadvantages such as poor specificity. Normal cells in the body lead to a series of side effects, such as hair loss, vomiting, nausea and loss of appetite, etc. Therefore, it is an urgent need to develop a class of drugs with high safety, small side effects, and good anti-tumor effects. Among them, polyunsaturated fatty acids Research and development has received widespread attention.

Fatty acids are the main components of fats, phospholipids and glycolipids, and are the material basis of life. According to whether its carbon chain contains unsaturated bonds or double bonds, it can be divided into saturated fatty acids and unsaturated fatty acids. Fatty acids containing more than two unsaturated bonds are called polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) contain five and six double bonds, respectively. According to its configuration, fatty acids are divided into cis (Z type) and trans (E type). EPA and DHA in common deep-sea fish oil are Z type, which are considered to be ω- Group 3 fatty acids. Studies have shown that Z-EPA and Z-DHA have important functions such as enhancing human immunity, anti-allergy and promoting intracellular fatty acid metabolism, and DHA also plays an important role in the development of the nervous system of infants. Since the 1970s, researchers have found that Z-EPA and Z-DHA have the ability to inhibit the growth of various tumor cells, such as the half lethal concentration (IC ₅₀ ) of the human promyelocytic acute leukemia cell line HL60, respectively. 85μM and 70μM, the IC ₅₀ of human breast cancer cell line MCF-7 is 85±5μM and 65±5μM respectively; their main mechanism of action is to induce tumor cell apoptosis or cause tumor cell growth cycle arrest. At present, because Z-EPA and Z-DHA must be in high concentration to produce a killing effect on various tumor cells, their application is greatly limited.

There are two types of trans (E-type) fatty acids, naturally occurring and artificially produced. For example, dairy products such as cow and goat milk contain 4-9% E-type fatty acids or esters, while hydrogenated oil is produced by hydrogenating vegetable oils. An unsaturated fatty acid containing 14.2-34.3% E-type fatty acid; conjugated linoleic acid (CLA) has an E-type carbon-carbon double bond and is also classified as E-type fatty acid. The biological functions of such fatty acids are controversial, especially in the two seemingly opposite aspects of carcinogenesis and cancer treatment. Some studies have shown that CLA can be used as an adjuvant therapy for breast cancer, and some studies have shown that it can accelerate the malignant process of breast cancer. Other studies have shown that there is no correlation between the two, so far no conclusion has been reached. The research on E-type fatty acids is mainly based on E-oleic acid. It is believed that high intake of E-oleic acid will increase the risk of cardiovascular diseases, especially coronary heart disease, but it has no effect on early growth and development, type Ⅱ diabetes, high The correlation between blood pressure, cancer and other diseases and trans fat is currently not confirmed by clear evidence. E-type EPA and DHA, as isomers of Z-type EPA and DHA, have not been reported so far.

Antarctic krill oil is rich in polyunsaturated fats, especially omega-3 unsaturated fatty acids, of which the content of EPA and DHA is as high as more than 20%. It is generally believed that EPA and DHA in Antarctic krill are both Z-type, which have anti-oxidation, anti-aging, anti-tumor effects, but in fact, they are mostly analogous and lack experimental basis. The present invention uses methods such as solvent extraction, silica gel chromatography, and preparation of high-performance liquid chromatography to separate and purify Antarctic krill oil, and identifies two single The components are E-type EPA and DHA, and its anti-tumor activity is better than Z-type-based commercially available fish oil EPA and DHA, which provides new clues for the development of new anti-tumor drugs.

Contents of the invention

The object of the present invention is to provide a class of antitumor agents with good antitumor effect, high safety and few side effects—all-trans polyunsaturated fatty acids, E-type EPA and DHA. Using mixed solvent extraction, silica gel chromatography, and preparative liquid chromatography to separate and purify the effective components with antitumor activity in Antarctic krill oil, through gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, high-resolution electrospray Instruments such as ionization mass spectrometry, nuclear magnetic resonance, and Raman spectroscopy were used to analyze the structure of the active ingredients, and their effects on the growth of various tumor cells were investigated, and compared with commercially available fish oil EPA and DHA and sturgeon liver EPA and DHA.

The invention provides an all-trans EPA or all-trans DHA with anti-tumor activity, which is isolated from Antarctic krill oil. The EPA and DHA in the common deep-sea fish oil are Z-type, and the E-type EPA and DHA are isomers of the Z-type EPA and DHA, so far no anti-tumor activity has been reported.

The present invention also provides a method for isolating natural all-trans EPA or all-trans DHA with antitumor activity from Antarctic krill oil, which includes the following steps:

(1) Antarctic krill oil was added to a silica gel column for column chromatography separation, the eluent was collected, and the solvent was removed to obtain Fraction I and Fraction II. Fraction I was a yellow oil, and Fraction II was a bright red oil;

(2) Fraction II is diluted with acetonitrile, separated by high-performance preparative liquid chromatography, eluted with a mixed solvent system of acetonitrile and water as the mobile phase, and the different components obtained by elution are collected, and the solvent is removed to obtain component II in sequence -1 to Ⅱ-5 are all colorless or slightly yellow oily substances, of which component Ⅱ-2 is all-trans EPA and component Ⅱ-4 is all-trans DHA.

Further, in the column chromatography separation described in step (1), the eluent is a mixture of n-hexane and ether with a volume ratio of 7:3-9:1, and the volume percentage added to the mixture is 1 -5% acetic acid.

Further, the particle size of the silica gel described in step (1) is 300-400 mesh, and the mass ratio of silica gel to Antarctic krill oil is 30-100:1, preferably 50-80:1, more preferably 60- 70:1, the height-to-diameter ratio of the chromatographic column is 10-16:1.

Further, the chromatographic column filler described in step (2) is reversed-phase C ₁₈ , with a particle size of 10 μm, and the elution procedure of the mobile phase is as follows: elute with 62-68% acetonitrile between 0-10 min; Between elution 10-20min, use 67-73% acetonitrile for elution; Between elution 20-60min, use 72-78% acetonitrile for elution, the elution procedure of more preferred mobile phase is as follows in turn: Elution 0- For 10 minutes, use 65% acetonitrile for elution; for 10-20 minutes, use 70% acetonitrile for elution; for 20-60 minutes, use 75% acetonitrile for elution.

In the above separation method, the Antarctic krill oil is loaded onto the silica gel column, and the eluent is used for elution, and the eluent is collected. The volume of each fraction is 1/20 to 1/40 of the retention volume of the chromatographic column, preferably It is 1/30, and at the same time, the substances of different polarities contained in the obtained eluent are detected by thin layer chromatography, and the eluents with the same polarity fractions are combined at a temperature of 20-40 ° C and a pressure of - The solvent was removed by rotary evaporation under the condition of 0.08～-0.1Mpa, and Fraction I and Fraction II were obtained respectively. Fraction I was a yellow oil, and Fraction II was a bright red oil. Antitumor activity test results showed that Fraction Ⅱ had strong antitumor activity, and Fraction Ⅱ was further separated by preparative high performance liquid chromatography.

In the above separation method, the fraction II obtained by silica gel column chromatography separation is further separated by reverse _C18 high performance preparative liquid chromatography, using a mixed solvent of acetonitrile and water as the mobile phase, and the flow rate of the mobile phase is not particularly limited. Those skilled in the art can select an appropriate flow rate of the mobile phase according to conventional techniques in the art, preferably 10-20 mL/min, more preferably 15 mL/min. Detect the absorbance value at 210nm in ultraviolet light, collect in sections according to the peak eluting time, rotate and increase the recovery of solvent under the conditions of temperature 20-40°C and pressure -0.08～-0.1Mpa, and obtain components Ⅱ-1 to Components in sequence Points II-5. The results of antitumor activity test showed that components Ⅱ-2 and Ⅱ-4 had strong antitumor activity. The structures of component II-2 and component II-4 were identified by HRESIMS, ¹ H-NMR, ¹³ C-NMR, laser Raman scattering spectroscopy, etc., and it was determined that component II-2 was all-trans EPA, and component II -4 is all-trans DHA.

In the present invention, the described Antarctic krill oil is prepared as follows: Antarctic krill frozen shrimp or Antarctic krill powder are used as raw materials, mixed solvent (n-hexane: ethanol=10:1, v/v) at room temperature After stirring for 2 hours, the organic solvent phase was concentrated by rotary evaporation to obtain Antarctic krill shrimp oil. In order to obtain more free EPA and DHA, Antarctic krill oil can be hydrolyzed by enzymatic hydrolysis. The specific enzymatic hydrolysis conditions are: 1.0g Antarctic krill oil, 1.0g deionized water and 20mg immobilized lipase (Novozym435) in 15mL centrifuge tube, and then reacted in a shaker at 30°C for 24 hours under light-shielded conditions. After the reaction, the enzymatic hydrolyzate was extracted with chloroform, and then separated and purified. Commercially available products can also be used for Antarctic krill oil.

The present invention also provides the application of the above-mentioned all-trans EPA or all-trans DHA in the preparation of antitumor drugs. Preferably, the tumor is human breast cancer, hepatocellular carcinoma, promyelocytic leukemia, chronic myeloid leukemia, histiocytic lymphoma or prostate cancer.

The present invention also provides a pharmaceutical composition comprising the above-mentioned all-trans EPA and a pharmaceutically acceptable auxiliary material, or comprising the above-mentioned all-trans DHA and a pharmaceutically acceptable auxiliary material. Preferably, the pharmaceutical composition is granules, tablets or capsules.

The present invention has the following beneficial effects:

The invention adopts the method of silica gel chromatography coupled with preparative liquid chromatography to separate all-trans EPA (E-EPA) or all-trans DHA (E-DHA) with antitumor activity from Antarctic krill oil. EPA and DHA in common deep-sea fish oil are Z-type, and E-type EPA and DHA are isomers of Z-type EPA and DHA, which have not been reported so far. E-EPA and E-DHA of Antarctic krill have excellent killing ability on various tumor cells, and their effects are stronger than EPA and DHA derived from commercially available fish oil and sturgeon liver oil, and have no obvious growth inhibition on normal liver cell HL7702, It has the potential to develop new antitumor drugs.

Description of drawings

Figure 1 is a thin-layer chromatogram representing fractions of different polarities;

Fig. 2 is the preparation high performance liquid phase chromatogram that represents silica gel chromatography fraction II;

Fig. 3 is to represent the growth inhibition curve of preparation component II-2 to MCF-7;

Fig. 4 is to represent the growth inhibition curve of preparation component II-4 to MCF-7;

Fig. 5 is the mass spectrogram representing component II-2 (EPA);

Fig. 6 is the mass spectrogram representing component II-4 (DHA);

Fig. 7 is a ¹ H-NMR chart showing component II-2 (EPA);

Fig. 8 is a ¹ H-NMR chart showing component II-4 (DHA);

Fig. 9 is a ¹³ C-NMR chart showing component II-2 (EPA);

Fig. 10 is a ¹³ C-NMR chart showing component II-4 (DHA);

Fig. 11 is the laser Raman scattering spectrogram representing Antarctic krill and commercially available fish oil EPA;

Fig. 12 is a laser Raman scattering spectrum diagram showing Antarctic krill and commercially available fish oil DHA.

Detailed ways

The following non-limiting examples can enable those skilled in the art to understand the present invention more fully, but do not limit the present invention in any way. In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical companies.

In the specific embodiment of the present invention, relate to following experimental technique and analytical method:

1. Cell Culture

Human breast cancer cell lines MCF-7 and MDA-MB-231, human liver cancer cell line SMMC-7721, human promyelocytic acute leukemia cell line HL60, human chronic myeloid leukemia cell line K562, human histiocytic lymphoma cell line U937, human prostate cancer cell line PC-3 and human normal liver tissue cells HL7702 were all purchased from the Cell Bank of the Chinese Academy of Sciences. The medium used for MCF-7 and MDA-MB-231 was DMEM high glucose medium (Hyclone), and the rest The medium used for the cells was RPMI-1640 medium (Hyclone), and 10% FBS (Hyclone) and 1% penicillin-streptomycin mixed solution (Hyclone) were added during the culture process. All cells ^were cultured in 25cm2 culture flasks. After the above cells were cultured in the culture flask for two days, the adherent cells and suspension cells were treated accordingly: the adherent cells were washed with PBS buffer, then a certain amount of trypsin solution was added, and then the corresponding medium was added and gently blown until the cells were dispersed. The number of cells was counted using a cell counting plate; the suspended cells were centrifuged at 800r/min and the supernatant was removed, washed twice with PBS buffer and resuspended with the corresponding medium, and then counted using a cell counting plate. The above cells were added to a 96-well plate, the number of cells in each well was controlled to be 3000, the volume of medium in each well was 200 μL, and the 96-well plate was placed in a cell culture incubator for 24 hours.

2. Detection of anti-tumor activity

Use MTT method or CCK-8 kit to detect the effect of isolated and purified substances on the growth of different tumor cells to determine their anti-tumor activity. Adherent cells were detected using the MTT method, and suspension cells were detected using the CCK-8 kit. The substance to be tested was dissolved in DMSO before detection, and the culture medium containing 0.5% DMSO was used as a control. First place the 96-well plate with the substance to be tested or the control solution at the appropriate concentration in a cell culture incubator and incubate for 16-18 hours, add 20 μL of MTT or CCK-8 (Kagen) reagent to each well, and incubate for another 4 hours. Absorbance was detected at 570nm (MTT)/450nm (CCK-8) and 630nm respectively. Cell viability was calculated according to the following formula:

Cell survival rate (MTT method, %)=[average value of absorbance value (A ₅₇₀ -A ₆₃₀ ) of experimental group/average value of absorbance value (A ₅₇₀ -A ₆₃₀ ) of control group]*100

Cell survival rate (CCK-8 method, %)=[average value of absorbance value (A ₄₅₀ -A ₆₃₀ ) of experimental group/average value of absorbance value (A ₄₅₀ -A ₆₃₀ ) of control group]*100

The IC ₅₀ of the median lethal concentration was calculated using the modified Cole's method formula:

lg(IC ₅₀ )=X _m -I*(P-(3-P _m -P _n )/4)

Among them, X _m is lg (maximum drug concentration), I is lg (maximum drug concentration/sub-highest drug concentration), P is the sum of the cell survival rate of the experimental group, P _m is the maximum cell survival rate, and P _n is the minimum cell survival rate .

3. Extraction of Antarctic krill oil:

First, use Antarctic krill frozen shrimp or Antarctic krill powder as raw materials, stir at room temperature for 2 hours with a mixed solvent (n-hexane: ethanol = 10:1, v/v), extract the shrimp oil in the raw material, and rotate the organic solvent phase Antarctic krill shrimp oil can be obtained after evaporation and concentration. Similarly, a mixture of solvents was used to extract oil from sturgeon liver.

The separation of various components in the Antarctic krill oil of embodiment 1

(1) Preparation of silica gel column: 300 g of activated 300-400 mesh silica gel was wet-packed into a glass separation column with an inner diameter of 3.0 cm and a length of 35.0 cm, and equilibrated with n-hexane.

(2) Take 5g of Antarctic krill shrimp oil, add it to the prepared silica gel column for column chromatography separation, and use eluent A (n-hexane: ethyl ether: acetic acid=80:20:1.5, v/v/v) ) and eluent B (chloroform:methanol:water=60:30:5, v/v/v), each fraction is 10mL, and the different poles of the substances contained in the eluent are detected by thin layer chromatography As shown in Figure 1, Antarctic krill oil is used as a comparison in the figure, and numbers i, ii, and iii correspond to triglycerides or substances whose polarity is close to triglycerides, oleic acid or substances whose polarity is close to oleic acid substances, phospholipids. Fractions with the same polarity were combined according to the results of thin-layer chromatography, and finally four groups of fractions were obtained, among which fraction I and fraction II were obtained by eluting with eluent A, and fraction III and fraction IV were obtained by eluting with eluent B. The total volumes of the collected four fractions were 510, 420, 285, and 225 mL respectively, and the fractions were concentrated by rotary evaporation for later use. After concentrated by vacuum rotary evaporation, the masses of the four fractions were 2.78, 1.12, and 0.59, respectively. and 0.17g. Among them, Fraction I is yellow oily substance, Fraction II is bright red oily substance, Fraction III and Fraction IV are dark red oily substance. The thin-layer chromatography conditions in Figure 1 are: use a silica gel thin-layer chromatography plate, lightly draw a spotting line with a pencil at a distance of 1 cm from the bottom of the chromatography plate, and spot the eluent collected from each receiving tube at equal distances. Chromatography on the sample line and placed in a chromatography cylinder containing a developer, the developer is eluent A or eluent B, the developer used in Figure 1(a), 1(b) and 1(c) It is eluent A, and the developer used in 1(d) is eluent B. Under the above conditions, a better detection effect can be achieved. After testing the anti-tumor activity of tumor cells, Fraction Ⅱ has growth inhibitory effect on human promyelocytic acute leukemia cell line HL60 and human histiocytic lymphoma cell line U937, and has no growth inhibitory effect on human normal liver cell HL7702. All three fractions had no obvious effect on the above cells.

(3) Purification of Fraction II

Dilute and mix the fraction II obtained in step (2) with acetonitrile at a volume ratio of 1:3, and then load it on a preparative high performance liquid chromatography column, wherein the chromatographic column is a YMC preparative column (YMC-Pack ODS-A, 20×250mm, particle size 10μm), 0.6mL for each injection, using acetonitrile-water system as the eluent, elution at a constant flow rate of 15mL/min, the elution program is: 0-10 minutes, with 65% Acetonitrile aqueous solution elution; 10-20 minutes, 70% acetonitrile aqueous solution elution; 20-60 minutes, 75% acetonitrile elution. The absorbance value at 210nm was detected by ultraviolet light, and its spectrum is shown in Figure 2. Collect in sections according to the peak elution time, and a total of 5 substances were obtained, which were labeled as II-1 to II-5 according to the peak elution time, and the solvent was removed by a rotary evaporator for later use. After diluting to an appropriate concentration with DMSO, the MTT method was used to detect the growth inhibitory effects of various substances on MCF-7. The test results showed that Ⅱ-2 and Ⅱ-4 had different growth inhibitory effects on MCF-7 (as shown in Figures 3 and 4 shown), while Ⅱ-1, Ⅱ-3 and Ⅱ-5 had no growth inhibitory effect on MCF-7. The results in Fig. 3 show that the growth inhibitory effect of component II-2 on MCF-7 increases with the increase of the concentration, and the inhibition rate reaches 59% at the concentration of 12.5 μg/mL. The results in Fig. 4 show that the growth inhibitory effect of component II-4 on MCF-7 increases with the increase of the concentration, and the inhibition rate reaches 71% at the concentration of 12.5 μg/mL.

(4) Structural identification of components Ⅱ-2 and Ⅱ-4

Components II-2 and II-4 obtained in step (3) were detected by HRESIMS, ¹ H-NMR and ¹³ C-NMR, and the results are shown in Figures 5-10. Figure 5-6 shows that the relative molecular masses of the mass spectrogram fragments of these two substances are 320.2570 and 346.2707 respectively, and it is speculated that they may be NH4 ⁺ peaks. After calculation, the actual relative molecular masses of these two substances are 302.2246 and 328.2402, which are respectively comparable to those of EPA and DHA. The relative molecular mass data are consistent; the ¹ H-NMR spectra of the two substances (Figure 7-8) are in good agreement with the ¹ H-NMR spectra of EPA and DHA in the literature, and the specific results are shown in Table 1; the ¹³ C of the two substances - The NMR spectrum (Fig. 9-10) is also consistent with the data reported in the literature, so it can be determined that components II-2 and II-4 are EPA and DHA in ω-3 polyunsaturated fatty acids, respectively.

Table 1. Determination results of ¹ H-NMR spectra of two effective components Ⅱ-2 and Ⅱ-4

The laser Raman scattering spectra of EPA and DHA in Antarctic krill oil and commercially available fish oil (Figure 11, 12) show that the Raman spectra of two different sources of EPA and DHA are at shifts of 1658cm ^-1 , 1671cm ^-1 , 1265cm ^{- 1} and 1303cm ^-1 , there are obvious differences in the four places, the first two displacements represent the Z-type carbon-carbon unsaturated double bond and the E-type unsaturated double bond, and the ratio of the latter two displacements [ν(1303cm ^-1 )/ν(1265cm ^{- 1} )], the larger the number of Z-type carbon-carbon double bonds in the sample. No obvious E-type unsaturated double bonds can be seen in the Raman scattering spectrum of EPA and DHA extracted from Antarctic krill oil, so the number of Z-type unsaturated carbon-carbon double bonds of EPA and DHA in Antarctic krill oil is It is lower than the detection limit of Raman scattering spectrum, so it can be considered that the EPA and DHA are all-trans EPA and DHA, while there are two unsaturated carbon-carbon double bonds of E and Z in the commercially available fish oil EPA and DHA, and the Z type Much more than the E-type.

Example 2 Detection of Antitumor Activity of Antarctic Krill Oil EPA and DHA

The E-type EPA and DHA isolated from Antarctic krill oil have inhibitory effects on the growth of various tumor cells, and their IC ₅₀ values are shown in Table 2, and these values are lower than the control standard EPA and DHA, sturgeon liver and The corresponding IC ₅₀ values of EPA and DHA derived from commercially available fish oil, and compared with many anti-tumor drugs currently available on the market, Antarctic krill EPA and DHA have no obvious toxicity to normal human liver cells HL7702, so they have relatively good application. It has great advantages and broad application prospects.

Claims (7)

1. a method for separating natural all-trans EPA or all-trans DHA with antitumor activity, comprising the steps of: (1) Antarctic krill oil was added to a silica gel column for column chromatography separation, the eluent was collected, and the solvent was removed to obtain Fraction I and Fraction II. Fraction I was a yellow oil, and Fraction II was a bright red oil; (2) Fraction II is diluted with acetonitrile, separated by high-performance preparative liquid chromatography, eluted with a mixed solvent system of acetonitrile and water as the mobile phase, and the different components obtained by elution are collected, and the solvent is removed to obtain component II in sequence -1 to II-5, wherein component II-2 is all-trans EPA, and component II-4 is all-trans DHA. 2. separation method according to claim 1, is characterized in that, in the described column chromatography separation of step (1), described eluent is the normal hexane that volume ratio is 7:3-9:1 and diethyl ether mixed solution, and 1-5% acetic acid by volume is added to the mixed solution. 3. separation method according to claim 1, is characterized in that, the particle diameter of the silica gel described in step (1) is 300～400 orders, and the mass ratio of silica gel and Antarctic krill oil is 30～100:1, The height-to-diameter ratio of the chromatographic column is 10-16:1. 4. separation method according to claim 1, it is characterized in that, the high-efficiency preparative liquid phase chromatographic column filler described in step (2) is reverse phase C ₁₈ , particle diameter 10 μ m, the elution program of mobile phase is as follows successively: For elution between 0-10min, use 62-68% acetonitrile; for 10-20min, use 67-73% acetonitrile; for 20-60min, use 72-78% acetonitrile . 5. the natural all-trans EPA that separation method as claimed in claim 1 obtains or the application of all-trans DHA in the preparation antitumor drug. 6. The application according to claim 5, wherein the tumor is human breast cancer, hepatocellular carcinoma, promyelocytic leukemia, chronic myeloid leukemia, histiocytic lymphoma or prostate cancer. 7. A pharmaceutical composition comprising the natural all-trans DHA obtained by the separation method as claimed in claim 1 and pharmaceutically acceptable auxiliary materials.