CN104815001A - Extraction and purification method for acquiring anti-tumor active ingredient substances from linseed oil - Google Patents
Extraction and purification method for acquiring anti-tumor active ingredient substances from linseed oil Download PDFInfo
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Abstract
The invention relates to an extraction and purification method for acquiring anti-tumor active ingredient substances from linseed oil. The extraction and purification method comprises the following steps: (1) placing an ethanol solution and linseed oil into a mixer to be mixed, regulating the pH, stirring, leaving to stand till a layered interface is formed, then performing rotary concentration to obtain an extract; (2) placing the extract obtained from the step (1) in an extraction tank, adding subcritical water, performing rotary concentration on an obtained extracting solution, and carrying out vacuum freeze-drying to obtain a crude extract; and (3) weighing the crude extract obtained in the step (2), and utilizing the high-speed counter-current chromatography to purify a solvent system to obtain a purified extract. In the extraction and purification process, a solvent capable of being applied to food production is adopted to conform to the relevant laws and regulations of China, and the risk of residues caused by application of lots of organic solvents is avoided. The tumor cell proliferation experiments testify that the obtained extract has the cytological effect of inhibiting the growth of tumor cells and can be applied to health food assisting in inhibiting tumors.
Description
Technical field
The present invention relates to effective ingredients in plant extraction and purification process, particularly relate to a kind of method for extraction and purification obtaining anti-tumor active ingredient material from Semen Lini oil, belong to plant processing extraction field.
Background technology
Semen Lini (Linum ustitatissimum L), also known as linum, belongs to flax family (Linaceae), linum, is one of large oil crop in the world ten, mainly for countries such as Canada, Argentina, India, the U.S., China.Main containing functional components such as fat, albumen, saccharides in Semen Lini, what is more important Semen Lini is rich in the Semen Lini oil of alpha-linolenic acid content, and Semen Lini oil is one of known plants oils and fats that current n-3 content of fatty acid is the highest.Recent study finds, Semen Lini oil has prevention hyperlipemia, atherosclerosis, reduces blood pressure, strengthens insulin and fall hypoglycemic effect.
Cancer is the large assailant threatening human health, is the second largest fatal disease (if by cardiovascular disease and cerebrovascular disease separately statistics, cancer is then positioned at first) being only second to cardiovascular and cerebrovascular disease, seriously threatens human health and life.Although the cancer therapy drug kind applied clinically is now many, expensive, toxic and side effects is serious, and does not still treat the specific drug of some cancer.Plant has synthesized the secondary metabolite of various configurations in very long evolutionary process, these secondary metabolites not only have different biological activitys, also usually be found to have new mechanism of action, find at interior occurring in nature from plant and find that the anticancer active constituent of high-efficiency low-toxicity or lead compound are the focuses of the problem extremely paid close attention to of domestic and international medicine scholar and research always.
At present, patent No. CN101945888A discloses a kind of method from recovery of oil hydrophobic peptide, adopts and extract oily substance from vegetable material, and by nonpolar for the separating of oil one-tenth of extraction and polarity fraction, Separation and Recovery hydrophobicity peptide matters from different fractions.The method have employed multiple organic solvent in a large number as hydrocarbon, ketone, ester, ether, aromatic substance etc. in implementation process, the use of these organic solvents, cause dissolvent residual in product, the composition obtained by the method cannot be applied in domestic daily health food, and experimentation conditional request is harsher, cause certain injury to staff's health, the technics comparing of the method is loaded down with trivial details, and the probability realizing large-scale production future is lower.
Summary of the invention
The present invention is directed to the deficiency that above-mentioned prior art exists, a kind of method for extraction and purification obtaining anti-tumor active ingredient material from Semen Lini oil is provided.
The technical scheme that the present invention solves the problems of the technologies described above is as follows: a kind of method for extraction and purification obtaining anti-tumor active ingredient material from Semen Lini oil, comprises the following steps:
(1) extract: alcoholic solution and Semen Lini oil are placed in mixed instrument and mix, regulate pH, stir, leave standstill after layering interfaces is formed, spin concentration, form extractum;
(2) Subcritical water chromotagraphy: the extractum of step (1) is positioned in extraction pot, adds subcritical water, by the extracting solution spin concentration obtained, vacuum lyophilization obtains crude extract;
(3) high speed adverse current chromatogram purification: take the crude extract that step (2) obtains, utilize high speed adverse current chromatogram purification solvent system, normal hexane, ethyl acetate, first alcohol and water are added in separatory funnel, concussion makes solution fully mix, place 1h, divide after balancing each other and separate upper and lower phase; Lower to immobile phase with solvent system, upper is mobile phase mutually, and adjustment rotating speed, pump into mobile phase, reclaim under reduced pressure reagent, obtains purified extract.
On the basis of technique scheme, the present invention can also do following improvement.
Further, the volume ratio of the alcoholic solution described in step (1) and Semen Lini oil is (1 ~ 5): 1, and the volumetric concentration of described alcoholic solution is 90 ~ 100%.
Further, in step (1), regulate pH to be 4.5 ~ 6, stir 0.5 ~ 1h.
Further, described in step (2), the weight ratio of extractum and subcritical water is 1:(10 ~ 50)
Further, described in step (2), cryogenic temperature is-20 ~-40 DEG C.
Further, the spin concentration described in step (2) refers to the liquid screwing out extracting solution 3/5 ~ 4/5 under 52 ~ 68 DEG C of conditions.
Further, in the extraction pot described in step (2), Extracting temperature is 50 ~ 120 DEG C, and extraction pressure is 3 ~ 5MPa, and extraction time is 10 ~ 40min, pressurizes 1 ~ 3 time.
Further, described in step (3), the volume ratio of normal hexane, ethyl acetate, first alcohol and water is (1 ~ 3): (2 ~ 4): (3 ~ 5): (1 ~ 3).
Further, the rotating speed described in step (3) is 600 ~ 800rpm, and the flow velocity pumping into mobile phase is 2 ~ 4ml/min.
The invention has the beneficial effects as follows: the present invention adopts alcoholic solution to extract and supertension coupling Subcritical water chromotagraphy technology obtains the Semen Lini oil crude extract that can be used for health food industry, and by high-speed countercurrent chromatography to crude extract purification.In extraction purification process, have employed the solvent that can be used for food production, meet the rules and regulations that country is relevant, avoid a large amount of organic solvents to use the residual risk caused.The crude extract obtained by said method and purified carry out the qualification of material aspect by mass spectrum, determine that this extract contains the cyclic peptide composition mentioned in document, and content are higher.Crude extract and purified are passed through tumor cell proliferation experimental verification, all there is cytology's effect of inhibition tumor cell growth, can be used for the application of auxiliary Tumor suppression health food aspect.
Accompanying drawing explanation
Fig. 1 is the mass spectrum that the embodiment of the present invention 1 obtains property material;
Fig. 2 is the total ion chromatogram that the embodiment of the present invention 1 obtains property material.
Detailed description of the invention
Be described principle of the present invention and feature below in conjunction with example, example, only for explaining the present invention, is not intended to limit scope of the present invention.
Embodiment 1
1000ml Semen Lini oil is injected mix and blend container, and adds 90% (v/v) alcoholic solution according to 1:1 ratio, adjust ph to 4.5, mixing time 0.5h, leave standstill, after layering interfaces is formed, be separated alcohol phase liquid, spin concentration becomes extractum; Gained extractum is positioned in sub critical extraction tank, inject subcritical water, lixiviate ratio is 1:20, Extracting temperature is 50 DEG C, extraction pressure is 3MPa, and extraction time is 10min, pressurizes 1 time, obtain extracting solution spin concentration, vacuum freezing-20 DEG C of dryings obtain the crude extract 15g being rich in cyclic peptide composition; Take crude extract 200mg, high speed adverse current chromatogram solvent system is normal hexane-ethyl acetate-Methanol-water is solvent system, solvent ratios is 1:3:3:1, upper is mobile phase mutually, lower is immobile phase mutually, and adjustment rotating speed 600rpm, enters mobile phase purification of crude extract with 3.0ml/min flow pump, by high performance liquid chromatography method, obtain cyclic peptide composition Secondary concentrate and be about 62mg.
The cyclic peptide Secondary concentrate obtained is carried out UPLC-MS analysis, as shown in Figure 1 and Figure 2.
Mass-spectrometric technique is a kind of authenticate technology, and it can measure the molecular weight of biomacromolecule fast and very exactly, thus can identification and analysis material fast.Fig. 1 spectrum in exist m/z 977.69 [M+H]+, m/z 999.99 [M+Na]+, above-mentioned two peaks, as the criterion of molecular weight of material, prove that its molecular weight is 996.69, with describe in interrelated data consistent.Illustrate, this Secondary concentrate contains this material of ring E.In addition, by other the spectrum peak occurred in total ion current (Fig. 2), according to its MS collection of illustrative plates, infer that other spectrum peaks in total ion are cyclic peptide series material or fragments of some polypeptide in Semen Lini oil.
The crude extract obtained by the present embodiment and purified are by tumor cell proliferation experimental verification.
1, cell strain and condition of culture
This external cancer cell for extract in Semen Lini oil is four kinds of conventional cancerous cell kinds such as hepatocellular carcinoma H22, external ovarian cancer cell HO8910pm, S180 tumor cell, Lewis lung sarcoma cell.With the RPMI RPMI-1640 containing 10% hyclone, 100U/m penicillin and streptomycin, in 37 DEG C, 5%CO
2secondary Culture in the incubator of saturated humidity, trophophase cell of taking the logarithm is tested.
2, cancer cell in vitro proliferation experiment
Mtt assay, also known as MTT colorimetry, is a kind of method detecting cell survival and growth.Its Cleaning Principle is that the succinate dehydrogenase in living cells mitochondrion can make exogenous MTT be reduced to water-insoluble bluish violet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and be deposited in cell, and dead cell is without this function.First a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) energy dissolved cell, measures its absorbance value with enzyme-linked immunosorbent assay instrument at 490nm wavelength place, can indirectly reflect living cells quantity.Within the scope of certain cell number, the amount that MTT crystallization is formed is directly proportional to cell number.The method has been widely used in Activity determination, large-scale screening anti-tumor medicine, cell toxicity test and the tumor radiosensitivity mensuration etc. of some bioactie agents.Its feature be highly sensitive, economy is strong.
The cell that phase of taking the logarithm grows, with every hole 5 × 10
3be inoculated in 96 well culture plates, culture plate divides into groups, and often group establishes 3 multiple holes.
1st group only adds cell culture fluid, as blank group;
2nd group adds cell culture fluid and cell suspension, is positioned over 37 DEG C, 5%CO
2cultivate 24 hours under condition, as the cell controls group not adding example pharmaceuticals;
3rd group adds cell culture fluid and cell suspension, is positioned over 37 DEG C, 5%CO
2cultivate after 24 hours under condition, add the sample effect liquid of variable concentrations, as medicine group; Medicine group final concentration is respectively 25 μ g/mL, 50 μ g/mL, 75 μ g/mL, 100 μ g/mL.
After 48h is cultivated in each group of continuation, every hole adds the MTT 20 μ L of 5mg/mL, after effect 4h, the centrifugal 8min of 1500rpm, removes supernatant, and every hole adds DMSO 150 μ L, shake 10 minutes, first a ceremonial jade-ladle, used in libation is thoroughly dissolved, and 492nm light-metering absorbs, and calculating variable concentrations and the sample of action time are to the suppression ratio of tumor cell proliferation.
3, experimental result
Embodiment 1 obtain crude extract to the suppression suppression ratio of different tumor cell line as following table:
Embodiment 1 obtain pure extract to the suppression suppression ratio of different tumor cell line as following table:
To sum up test proof, the present invention extracts the material obtained from Semen Lini oil all has obvious inhibitory action to the propagation of several tumor cell, and extract concentrations is higher, also improves the suppression ratio of cancerous cell thereupon, and plays potential anti-tumor activity further.Adopt method of the present invention to extract the bitter substance in Semen Lini oil, make said multiple dosage form on pharmaceutics, for the preparation of the functional food can assisting inhibition tumor cell effect, the dosage form of functional food can be the form such as capsule-type, tablet.
Embodiment 2
1000ml Semen Lini oil is injected mix and blend container, and adds 95% (v/v) alcoholic solution according to 1:3 ratio, adjust ph to 5, mixing time 0.8h, leave standstill, after layering interfaces is formed, be separated alcohol phase liquid, spin concentration becomes extractum; Be positioned over by gained extractum in sub critical extraction filling, inject subcritical water, lixiviate ratio is 1:30, Extracting temperature is 70 DEG C, and extraction pressure is 4MPa, and extraction time is 20min, pressurize 2 times, obtain extracting solution spin concentration, vacuum freezing-30 DEG C of dryings obtain being rich in cyclic peptide composition crude extract 24g; Take crude extract 200mg, high speed adverse current chromatogram solvent system is normal hexane-ethyl acetate-Methanol-water is solvent system, solvent ratios is 2:4:5:1, upper is mobile phase mutually, lower is immobile phase mutually, and adjustment rotating speed 700rpm, enters mobile phase purification of crude extract with 2.0ml/min flow pump, by high performance liquid chromatography method, obtain cyclic peptide composition Secondary concentrate and be about 59mg.
Embodiment 3
1000ml Semen Lini oil is injected mix and blend container, and adds 96% (v/v) alcoholic solution according to 1:5 ratio, adjust ph to 6, mixing time 1h, leave standstill, after layering interfaces is formed, be separated alcohol phase liquid, spin concentration becomes extractum; Be positioned over by gained extractum in sub critical extraction filling, inject subcritical water, lixiviate ratio is 1:40, Extracting temperature is 100 DEG C, and extraction pressure is 5MPa, and extraction time is 30min, pressurize 3 times, obtain extracting solution spin concentration, vacuum freezing-20 DEG C of dryings obtain being rich in cyclic peptide composition crude extract 31g; Take crude extract 200mg, high speed adverse current chromatogram solvent system is normal hexane-ethyl acetate-Methanol-water is solvent system, solvent ratios is 3:2:3:2, upper is mobile phase mutually, lower is immobile phase mutually, and adjustment rotating speed 800rpm, enters mobile phase purification of crude extract with 4.0ml/min flow pump, by high performance liquid chromatography method, obtain cyclic peptide composition Secondary concentrate and be about 70mg.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. from Semen Lini oil, obtain a method for extraction and purification for anti-tumor active ingredient material, comprise the following steps:
(1) extract: alcoholic solution and Semen Lini oil are placed in mixed instrument and mix, regulate pH, stir, leave standstill after layering interfaces is formed, spin concentration, form extractum;
(2) Subcritical water chromotagraphy: the extractum of step (1) is positioned in extraction pot, adds subcritical water, by the extracting solution spin concentration obtained, vacuum lyophilization obtains crude extract;
(3) high speed adverse current chromatogram purification: take the crude extract that step (2) obtains, utilize high speed adverse current chromatogram purification solvent system, normal hexane, ethyl acetate, first alcohol and water are added in separatory funnel, concussion makes solution fully mix, place 1h, divide after balancing each other and separate upper and lower phase; Lower to immobile phase with solvent system, upper is mobile phase mutually, and adjustment rotating speed, pump into mobile phase, reclaim under reduced pressure reagent, obtains purified extract.
2. method for extraction and purification according to claim 1, is characterized in that, the volume ratio of the alcoholic solution described in step (1) and Semen Lini oil is (1 ~ 5): 1, and the volumetric concentration of described alcoholic solution is 90 ~ 100%.
3. method for extraction and purification according to claim 1, is characterized in that, regulates pH to be 4.5 ~ 6 in step (1), stirs 0.5 ~ 1h.
4. method for extraction and purification according to claim 1, is characterized in that, described in step (2), the weight ratio of extractum and subcritical water is 1:(10 ~ 50).
5. method for extraction and purification according to claim 1, is characterized in that, described in step (2), cryogenic temperature is-20 ~-40 DEG C.
6. method for extraction and purification according to claim 1, is characterized in that, the spin concentration described in step (2) refers to the liquid screwing out extracting solution 3/5 ~ 4/5 under 52 ~ 68 DEG C of conditions.
7. method for extraction and purification according to claim 1, is characterized in that, in the extraction pot described in step (2), Extracting temperature is 50 ~ 120 DEG C, and extraction pressure is 3 ~ 5MPa, and extraction time is 10 ~ 40min, pressurizes 1 ~ 3 time.
8. method for extraction and purification according to claim 1, it is characterized in that, described in step (3), the volume ratio of normal hexane, ethyl acetate, first alcohol and water is (1 ~ 3): (2 ~ 4): (3 ~ 5): (1 ~ 3).
9. method for extraction and purification according to claim 1, is characterized in that, the rotating speed described in step (3) is 600 ~ 800rpm, and the flow velocity pumping into mobile phase is 2 ~ 4ml/min.
10. method for extraction and purification described in the arbitrary claim of claim 1-9 is in the application of Semen Lini oil manufacture field.
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Citations (4)
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|---|---|---|---|---|
| CN1690076A (en) * | 2004-04-23 | 2005-11-02 | 中国科学院海洋研究所 | Use of ocean green algae in extraction of cyclic peptide components with anti-tumor activity |
| CN101945888A (en) * | 2007-12-21 | 2011-01-12 | 萨斯喀彻温大学 | Recovery of hydrophobic peptides from oils |
| CN102925286A (en) * | 2012-10-28 | 2013-02-13 | 神池县粮油机械研究所 | Linseed oil debittering method and device |
| CN103222938A (en) * | 2012-01-31 | 2013-07-31 | 株式会社爱茉莉太平洋 | Skin external composition containing sugar apple seed extract |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1690076A (en) * | 2004-04-23 | 2005-11-02 | 中国科学院海洋研究所 | Use of ocean green algae in extraction of cyclic peptide components with anti-tumor activity |
| CN101945888A (en) * | 2007-12-21 | 2011-01-12 | 萨斯喀彻温大学 | Recovery of hydrophobic peptides from oils |
| CN103222938A (en) * | 2012-01-31 | 2013-07-31 | 株式会社爱茉莉太平洋 | Skin external composition containing sugar apple seed extract |
| CN102925286A (en) * | 2012-10-28 | 2013-02-13 | 神池县粮油机械研究所 | Linseed oil debittering method and device |
Non-Patent Citations (3)
| Title |
|---|
| GUI B ET AL: "Distribution of cyclolinopeptides in flaxseed fractions and products", 《J.AGRIC.FOOD CHEM》 * |
| 孙浩冉等: "脱脂亚麻籽中一种环肽类成分的研究", 《安徽农业科学》 * |
| 梁光义: "《中药化学》", 31 August 2005, 中医古籍出版社 * |
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