CN104815001B - A kind of method for extraction and purification obtaining anti-tumor active ingredient substance from linseed oil - Google Patents
A kind of method for extraction and purification obtaining anti-tumor active ingredient substance from linseed oil Download PDFInfo
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Abstract
The present invention relates to a kind of to obtain the method for extraction and purification of anti-tumor active ingredient substance from linseed oil, and steps are as follows: (1) being placed in mixed instrument ethanol solution with linseed oil and mix, adjust pH, stirring, it stands after layering interfaces are formed, spin concentration, forms medicinal extract；(2) medicinal extract of step (1) is placed in extractor, subcritical water, the extracting solution spin concentration that will be obtained is added, vacuum freeze drying obtains crude extract；(3) it weighs the crude extract that step (2) obtains and obtains purified extract using high speed adverse current chromatogram purification solvent system.The present invention, using the solvent that can be used for food production, meets the relevant rules and regulations of country, avoids remaining risk caused by a large amount of organic solvent use during extraction purification.By extract obtained by the above method by tumor cell proliferation experimental verification, the cytology effect for inhibiting growth of tumour cell is all had, can be used for assisting inhibiting the application in terms of tumor health food.
Description
Technical field
The present invention relates to effective ingredients in plant extraction and purification process, more particularly to it is a kind of obtained from linseed oil it is antitumor
The method for extraction and purification of active ingredient substance belongs to plant processing extraction field.
Background technique
Linseed (Linum ustitatissimum L) is also known as flax, belongs to flax family (Linaceae), linum, is the big oil plant in the world ten
One of crop, primarily for countries such as Canada, Argentina, India, the U.S., China.Fat, egg are mainly contained in linseed
The functional components such as white, carbohydrate, more importantly linseed is rich in the linseed oil of alpha-linolenic acid content, linseed oil is
One of highest known plants grease of 3 content of fatty acid of n-at present.Recent study discovery, linseed oil have prevention high in fat
Mass formed by blood stasis, the effect for atherosclerosis, reducing blood pressure, enhancing insulin reduction blood glucose.
Cancer is to threaten a big assailant of human health, is the second largest fatal disease for being only second to cardiovascular and cerebrovascular disease
If (cardiovascular disease and cranial vascular disease are separately counted, cancer if be located at first), seriously threaten human health and
Life.Although the anticancer drug type clinically applied now is many, expensive, toxic side effect is serious, and does not control still
Treat the specific drug of certain cancers.The secondary metabolism that plant has synthesized various configurations in very long evolutionary process produces
Object, these secondary metabolites not only have different bioactivity, are also usually found to have new mechanism of action, exist from plant
The anticancer active constituent or lead compound that discovery high-efficiency low-toxicity is found in interior nature are always domestic and international drug scholar ten
Divide the project of concern and the hot spot of research.
Currently, patent No. CN101945888A discloses a kind of method from oil recycling hydrophobic peptide, using from plant material
Oily substance is extracted in material, and the oil of extraction is separated into non-polar and polar fraction, is separated and recovered from different fractions hydrophobic
Property peptide matters.This method largely uses a variety of organic solvents such as hydrocarbon, ketone, ester, ether, aromatic substance etc. in implementation process,
The use of these organic solvents causes dissolvent residual in product, and the ingredient obtained by the method can not be applied to domestic day
In normal health food, and the requirement of experimentation condition is harsher, and certain injury, the party are caused to staff's body
A possibility that technics comparing of method is cumbersome, and future realizes large-scale production is lower.
Summary of the invention
The present invention in view of the deficiency of the prior art, provide it is a kind of obtained from linseed oil anti-tumor activity at
Divide the method for extraction and purification of substance.
The technical scheme to solve the above technical problems is that it is a kind of obtained from linseed oil anti-tumor activity at
Divide the method for extraction and purification of substance, comprising the following steps:
(1) it extracts: ethanol solution being placed in mixed instrument with linseed oil and is mixed, adjust pH, stirring stands boundary to be layered
After face is formed, spin concentration forms medicinal extract；
(2) Subcritical water chromotagraphy: the medicinal extract of step (1) is placed in extractor, and subcritical water is added, mentions what is obtained
Liquid spin concentration is taken, vacuum freeze drying obtains crude extract；
(3) high speed adverse current chromatogram purifies: weighing the crude extract that step (2) obtains, utilizes high speed adverse current chromatogram purification solvent
N-hexane, ethyl acetate, first alcohol and water are added in separatory funnel system, and concussion is sufficiently mixed solution, places 1h, split-phase
Upper and lower phase is separated after balance；Using the lower phase of solvent system as stationary phase, upper phase is mobile phase, adjusts revolving speed, is pumped into flowing
Reagent is recovered under reduced pressure in phase, obtains purified extract.
Based on the above technical solution, the present invention can also be improved as follows.
Further, the volume ratio of ethanol solution described in step (1) and linseed oil is (1~5): 1, the ethyl alcohol is molten
The volumetric concentration of liquid is 90~100%.
Further, it is 4.5~6 that pH is adjusted in step (1), stirs 0.5~1h.
Further, the weight ratio of medicinal extract described in step (2) and subcritical water is 1:(10~50)
Further, cryogenic temperature described in step (2) is -20~-40 DEG C.
Further, spin concentration described in step (2) refers to the back-out extracting solution 3/5~4/5 under the conditions of 52~68 DEG C
Liquid.
Further, Extracting temperature is 50~120 DEG C in extractor described in step (2), and extraction pressure is 3~5MPa,
Extraction time is 10~40min, is pressurizeed 1~3 time.
Further, n-hexane described in step (3), ethyl acetate, first alcohol and water volume ratio be (1~3): (2~4):
(3~5): (1~3).
Further, revolving speed described in step (3) is 600~800rpm, and the flow velocity for being pumped into mobile phase is 2~4ml/min.
The beneficial effects of the present invention are: the present invention is coupled Subcritical water chromotagraphy technology using ethanol solution extraction and super-pressure
The linseed oil crude extract that can be used for health food industry is obtained, and crude extract is purified by high-speed countercurrent chromatography.?
During extraction purification, using the solvent that can be used for food production, meets the relevant rules and regulations of country, avoid largely having
Risk is remained caused by solvent use.In terms of crude extract obtained by the above method and purified are carried out substance by mass spectrum
Identification, determines that the extract contains the cyclic peptide constituents mentioned in document, and content is higher.Crude extract and purified are passed through swollen
Tumor cell proliferation experimental verification all has the cytology effect for inhibiting growth of tumour cell, can be used for assisting inhibiting tumor health
Application in terms of food.
Detailed description of the invention
Fig. 1 is the mass spectrogram that property material is made in the embodiment of the present invention 1；
Fig. 2 is the total ion chromatogram that property material is made in the embodiment of the present invention 1.
Specific embodiment
Principles and features of the present invention are described below in conjunction with example, the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the invention.
Embodiment 1
1000ml linseed oil is injected and is mixed in container, and is molten according to 1:1 ratio 90% (v/v) ethyl alcohol of addition
Liquid adjusts pH value to 4.5, mixing time 0.5h, stands, after layering interfaces are formed, separate alcohol phase liquid, spin concentration is at leaching
Cream；Gained medicinal extract is placed in sub-critical extraction tank, subcritical water is injected, extraction ratio is 1:20, and Extracting temperature is 50 DEG C,
Extraction pressure is 3MPa, extraction time 10min, pressurizes 1 time, obtains extracting solution spin concentration, and -20 DEG C of vacuum refrigeration dry
To the crude extract 15g for being rich in cyclic peptide constituents；Crude extract 200mg is weighed, high speed adverse current chromatogram solvent system is n-hexane-second
Acetoacetic ester-Methanol-water is solvent system, and solvent ratios 1:3:3:1, upper phase is mobile phase, and lower phase is stationary phase, and adjustment turns
Fast 600rpm enters mobile phase purifying crude extract with 3.0ml/min flow pump, by high performance liquid chromatography method, obtain cyclic peptide at
Enriched substance about 62mg in two times.
Obtained cyclic peptide Secondary concentrate is subjected to UPLC-MS analysis, as shown in Figure 1 and Figure 2.
Mass-spectrometric technique is a kind of identification technology, it can quickly and extremely accurately measure the molecular weight of large biological molecule,
So as to quick identification and analysis substance.Fig. 1 spectrum in there are m/z 977.69 [M+H]+, m/z 999.99 [M+Na]+, it is above-mentioned
Criterion of two peaks as molecular weight of material, it was demonstrated that its molecular weight is 996.69, consistent with described in relevant information.It says
Bright, this Secondary concentrate contains this substance of ring E.In addition, by the other spectral peaks occurred in total ion current (Fig. 2), according to
Its MS map, thus it is speculated that other spectral peaks in total ion are the fragments of cyclic peptide series material or some polypeptides in linseed oil.
The crude extract and purified that the present embodiment is obtained pass through tumor cell proliferation experimental verification.
1, cell strain and condition of culture
It is thin for hepatocellular carcinoma H22, external oophoroma that this is directed to external cancer cell system of extract in linseed oil
Four kinds of born of the same parents HO8910pm, S180 tumour cell, Lewis lung sarcoma cell etc. conventional cancer cell types.With contain 10% tire ox blood
Clearly, the RPMI 1640 culture medium of 100U/m penicillin and streptomysin, in 37 DEG C, 5%CO2It is passed in the incubator of saturated humidity
Culture, logarithmic growth phase cell are tested.
2, cancer cell in vitro proliferation experiment
Mtt assay is also known as MTT colorimetric method, is a kind of method for detecting cell survival and growth.Its testing principle is living cells
Succinate dehydrogenase in mitochondria can make exogenous MTT be reduced to bluish violet crystallization first a ceremonial jade-ladle, used in libation (Formazan) of water-insoluble simultaneously
It is deposited in cell, and dead cell is without this function.Dimethyl sulfoxide (DMSO) can dissolve the first a ceremonial jade-ladle, used in libation in cell, be examined with enzyme linked immunological
It surveys instrument and measures its absorbance value at 490nm wavelength, can reflect living cells quantity indirectly.Within the scope of certain cell number, MTT knot
The amount that crystalline substance is formed is directly proportional to cell number.This method is widely used in the Activity determination, large-scale of some bioactie agents
Screening anti-tumor medicine, cell toxicity test and tumor radiosensitivity measurement etc..Its feature is high sensitivity, economy
By force.
The cell for taking logarithmic phase to grow, with every hole 5 × 10396 well culture plates are inoculated in, culture plate grouping, every group sets 3
Multiple holes.
1st group is only added cell culture fluid, as blank control group；
2nd group of addition cell culture fluid and cell suspension, are placed in 37 DEG C, 5%CO2Under the conditions of cultivate 24 hours, as
The cell controls group of example pharmaceuticals is not added；
3rd group of addition cell culture fluid and cell suspension, are placed in 37 DEG C, 5%CO2Under the conditions of cultivate 24 hours after, add
The sample effect liquid for entering various concentration, as medicine group；Medicine group final concentration be respectively 25 μ g/mL, 50 μ g/mL, 75 μ g/mL,
100μg/mL。
Each group continues after cultivating 48h, and the 20 μ L of MTT of 5mg/mL is added in every hole, and after acting on 4h, 1500rpm is centrifuged 8min,
Supernatant is removed, 150 μ L of DMSO is added in every hole, shakes 10 minutes, dissolves first a ceremonial jade-ladle, used in libation thoroughly, and 492nm surveys light absorption, calculates different dense
Spend the inhibiting rate with the sample of action time to tumor cell proliferation.
3, experimental result
The crude extract that embodiment 1 obtains is as follows: the inhibition inhibiting rate of different tumor cell lines
The pure extract that embodiment 1 obtains is as follows: the inhibition inhibiting rate of different tumor cell lines
To sum up it is demonstrated experimentally that the substance that the present invention is extracted from linseed oil has the proliferation of several tumour cells
Apparent inhibiting effect, and extract concentrations are higher, also increase accordingly to the inhibiting rate of cancer cell, and further play potential
Anti-tumor activity.Bitter substance in linseed oil is extracted using method of the invention, is made described in pharmacy
A variety of dosage forms, are used to prepare the functional food that can assist inhibiting tumour cell effect, the dosage form of functional food can for capsule-type,
The forms such as tablet.
Embodiment 2
1000ml linseed oil is injected and is mixed in container, and is molten according to 1:3 ratio 95% (v/v) ethyl alcohol of addition
Liquid adjusts pH value to 5, mixing time 0.8h, stands, after layering interfaces are formed, separate alcohol phase liquid, spin concentration is at leaching
Cream；Gained medicinal extract is placed in sub-critical extraction filling, subcritical water is injected, extraction ratio is 1:30, and Extracting temperature is 70 DEG C,
Extraction pressure is 4MPa, extraction time 20min, pressurizes 2 times, obtains extracting solution spin concentration, and -30 DEG C of vacuum refrigeration dry
To rich in cyclic peptide constituents crude extract 24g；Crude extract 200mg is weighed, high speed adverse current chromatogram solvent system is n-hexane-acetic acid
Ethyl ester-Methanol-water is solvent system, and solvent ratios 2:4:5:1, upper phase is mobile phase, and lower phase is stationary phase, adjusts revolving speed
700rpm enters mobile phase purifying crude extract with 2.0ml/min flow pump and obtains cyclic peptide constituents by high performance liquid chromatography method
Secondary concentrate about 59mg.
Embodiment 3
1000ml linseed oil is injected and is mixed in container, and is molten according to 1:5 ratio 96% (v/v) ethyl alcohol of addition
Liquid adjusts pH value to 6, mixing time 1h, stands, after layering interfaces are formed, separate alcohol phase liquid, spin concentration is at medicinal extract；
Gained medicinal extract is placed in sub-critical extraction filling, subcritical water is injected, extraction ratio is 1:40, and Extracting temperature is 100 DEG C, is mentioned
Pressure power is 5MPa, extraction time 30min, pressurizes 3 times, obtains extracting solution spin concentration, -20 DEG C of vacuum refrigeration are dried to obtain
Rich in cyclic peptide constituents crude extract 31g；Crude extract 200mg is weighed, high speed adverse current chromatogram solvent system is n-hexane-acetic acid second
Ester-Methanol-water is solvent system, and solvent ratios 3:2:3:2, upper phase is mobile phase, and lower phase is stationary phase, adjusts revolving speed
800rpm enters mobile phase purifying crude extract with 4.0ml/min flow pump and obtains cyclic peptide constituents by high performance liquid chromatography method
Secondary concentrate about 70mg.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (6)
1. a kind of method for extraction and purification for obtaining anti-tumor active ingredient substance from linseed oil, comprising the following steps:
(1) it extracts: ethanol solution being placed in mixed instrument with linseed oil and is mixed, adjust pH, stirring is stood to layering interfaces shape
Cheng Hou, separation alcohol phase liquid carry out spin concentration, form medicinal extract；
(2) Subcritical water chromotagraphy: the medicinal extract of step (1) is placed in extractor, and subcritical water, the extracting solution that will be obtained is added
Spin concentration, vacuum freeze drying obtain crude extract；
(3) high speed adverse current chromatogram purifies: the crude extract that step (2) obtains is weighed, using high speed adverse current chromatogram purification solvent system,
N-hexane, ethyl acetate, first alcohol and water are added in separatory funnel, concussion is sufficiently mixed solution, 1h is placed, after split-phase balances
Separate upper and lower phase；Using the lower phase of solvent system as stationary phase, upper phase is mobile phase, adjusts revolving speed, is pumped into mobile phase, is depressurized
Reclaim reagent obtains purified extract；
The volume ratio of ethanol solution described in step (1) and linseed oil is (1~5): 1, the volume of the ethanol solution is dense
Degree is 90~100%；
It is 4.5~6 that pH is adjusted in step (1), stirs 0.5~1h；
Extracting temperature is 50~120 DEG C in extractor described in step (2), and extraction pressure is 3~5MPa, extraction time 10
~40min pressurizes 1~3 time；
N-hexane described in step (3), ethyl acetate, first alcohol and water volume ratio be (1~3): (2~4): (3~5): (1~
3)。
2. method for extraction and purification according to claim 1, which is characterized in that medicinal extract and subcritical water described in step (2)
Weight ratio be 1:(10~50).
3. method for extraction and purification according to claim 1, which is characterized in that cryogenic temperature described in step (2) is -20
~-40 DEG C.
4. method for extraction and purification according to claim 1, which is characterized in that spin concentration described in step (2) refers to
The liquid of extracting solution 3/5~4/5 is screwed out under the conditions of 52~68 DEG C.
5. method for extraction and purification according to claim 1, which is characterized in that revolving speed described in step (3) be 600~
800rpm, the flow velocity for being pumped into mobile phase is 2~4mL/min.
6. method for extraction and purification described in claim 1-5 any claim is in the application of linseed oil manufacture field.
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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 |
| 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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2015
- 2015-04-15 CN CN201510178126.2A patent/CN104815001B/en active Active
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 (2)
| Title |
|---|
| Distribution of cyclolinopeptides in flaxseed fractions and products;Gui B et al;《J.Agric.Food Chem》;20121231;第60卷(第35期);第8580-8589页 * |
| 脱脂亚麻籽中一种环肽类成分的研究;孙浩冉等;《安徽农业科学》;20101231;第38卷(第4期);第1693-1694页 * |
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