CN104116767B - A kind of preparation method of Alternanthera philoxeroides ligroin extraction and utilization - Google Patents
A kind of preparation method of Alternanthera philoxeroides ligroin extraction and utilization Download PDFInfo
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- CN104116767B CN104116767B CN201410313978.3A CN201410313978A CN104116767B CN 104116767 B CN104116767 B CN 104116767B CN 201410313978 A CN201410313978 A CN 201410313978A CN 104116767 B CN104116767 B CN 104116767B
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Abstract
The present invention relates to a kind of preparation method and application of Alternanthera philoxeroides ligroin extraction, belong to pharmaceutical technology field.The method of the invention is using Alternanthera philoxeroides as raw material, successively using techniques such as ultrasonic solvent extraction method, solvent extractions, and combine the drying means such as the dry, vacuum drying that is concentrated under reduced pressure, normal pressure concentrate drying, it is prepared into Alternanthera philoxeroides ligroin extraction, using the Alternanthera philoxeroides ligroin extraction being made as anticancer active constituent, it is allowed to be applied in treatment of cancer.This Alternanthera philoxeroides larger to environmental hazard of the present invention provides a kind of new approach, certain experiment basis and a kind of new thinking also are provided to develop the Anticancer Activities of Chinese herbal medicine simultaneously, active anticancer screening is carried out from the different extracts of Chinese herbal medicine.
Description
Technical field
The present invention relates to a kind of preparation method of Alternanthera philoxeroides ligroin extraction and utilization, belong to medical science neck
Domain.
Background technology
Alternanthera philoxeroides, alias alternanthera philoxeroides, revolution grass, water peanut etc., it is a kind of plant of Amaranthaceae.This plant is former
Production Brazil, nineteen thirty incoming China, be listed in Chinese first batch of alien species.The plant life power is extremely strong, adapts to aquatic and terrestrial
Environment, growth and breeding is rapid, and artificial introduces a fine variety diffusion and it is spread, and has become the pernicious of China's most area
Weeds, therefore the plant is also considered as a kind of very harmful invasive species.Studies have found that, the plant contains anthraquinone
Class, sterols, flavonoids, triterpenes etc. compound.Modern pharmacology research shows that the plant is clearing heat and detoxicating, cool blood, can use
The disease caused in the infection for the treatment of virus, and the disease such as Japanese Type-B encephalitis early stage, Hemorrhagic fever initial stage, measles.
But, so far, the research to the antitumaous effect of Alternanthera philoxeroides is also fewer, wherein TaiWan, China in 2007
The golden silks in side etc. the research of antitumaous effect has been carried out to the ethyl acetate extract of Alternanthera philoxeroides, it is found that it is thin to human cervical carcinoma
Born of the same parents Hela cells and l cell L929 have carried out antitumaous effect research, it is found that the ethyl acetate of Alternanthera philoxeroides is carried
Take thing that there is certain bioactivity to both cells.
Cancer is that the today's society incidence of disease is high, a kind of high persistent ailment of the death rate, not yet have at present can cure rate it is higher
Medicine is reported.The present inventor during herbal medicine anticancer active constituent, it is found that the petroleum ether of Alternanthera philoxeroides is extracted upon the look
Thing has good antitumor activity.This has established one for anticancer or the auxiliary therapeutic action research of this Plant Extracts of later stage
Fixed basis, also serves certain impetus to developing this invasive species for having harm.
The content of the invention
It is that Alternanthera philoxeroides exists it is an object of the invention to provide a kind of preparation method of Alternanthera philoxeroides ligroin extraction
Certain basis has been established in application in cancer therapy drug research, while being provided for this invasive species for having harm of the comprehensive regulation
Certain approach.
The preparation method of Alternanthera philoxeroides ligroin extraction of the present invention is used successively using Alternanthera philoxeroides as raw material
The techniques such as ultrasonic solvent extraction method, solvent extraction, and it is dry to combine the dry, vacuum drying that is concentrated under reduced pressure, normal pressure concentrate drying etc.
Drying method, is prepared into Alternanthera philoxeroides ligroin extraction, and the Alternanthera philoxeroides ligroin extraction being made is lived as anticancer
Property composition, is allowed to be applied in treatment of cancer.
The preparation method of Alternanthera philoxeroides ligroin extraction of the present invention specifically includes following steps：
（1）It is 1 by Alternanthera philoxeroides and ethanol volume ratio:2~1:5 ratio immerses Alternanthera philoxeroides in ethanol solution,
By ultrasonic extraction, extract to ethanol solution paler colour or it is colourless untill；
（2）Extract solution is collected, EtOH Sonicate extract is concentrated to without alcohol taste using vacuum distillation；
（3）Obtained EtOH Sonicate extract is collected, by the volume ratio 1 of sonicated extract and distilled water:1~1:2, super
Distilled water is added in sound extract and is uniformly mixed so as to obtain mixed solution, is 1 by the volume ratio of mixed solution and petroleum ether:1~1:2 ratio
Example adds petroleum ether in mixed solution and repeatedly extracted, until petroleum ether extraction is mutually colourless；
（4）Collect after petroleum ether extract, vacuum distillation concentration extract, extract absolute ethyl alcohol dissolves, Ran Houjin
Petroleum ether extract after the preliminary constant pressure and dry of row, preliminarily dried obtains drying petroleum ether extract after being dried in vacuo.
Alternanthera philoxeroides of the present invention for fresh collection or by dry rhizome, branches and leaves, one kind in fruit or
It is a variety of.
Step of the present invention（2）Described in vacuum distillation select Rotary Evaporators, condition is 43 DEG C ~ 48 DEG C, and vacuum is
0.04~0.06Mpa。
Step of the present invention（4）Described in vacuum distillation select Rotary Evaporators, condition is 43 DEG C ~ 48 DEG C, and vacuum is
0.04~0.06Mpa。
Step of the present invention（4）Described in constant pressure and dry during, drying temperature be 40 DEG C ~ 50 DEG C, drying time be 8 ~
In 12h, process of vacuum drying, drying temperature is 40 DEG C ~ 50 DEG C, and drying time is 8 ~ 12h, and vacuum is 0.06 ~ 0.08Mpa.
The mass percent concentration of ethanol of the present invention is 90% ~ 98%.
Utilization of the Alternanthera philoxeroides ligroin extraction of the present invention in cancer therapy drug is prepared.
The ligroin extraction preparation technology of Alternanthera philoxeroides is simple and practical, while having good active anticancer.To be comprehensive
Close and provide a kind of new approach using this invasive species larger to environmental hazard, while the also anticancer for exploitation Chinese herbal medicine
It is that the different extracts for selecting Chinese herbal medicine carry out anticancer that activity research, which provides certain experiment basis and a kind of new thinking,
Screening active ingredients.
Brief description of the drawings
The Alternanthera philoxeroides ligroin extraction of Fig. 1 embodiments 1 suppresses MCF-7 cell growth time effect curves；
The Alternanthera philoxeroides ligroin extraction of Fig. 2 embodiments 1 suppresses A549 cell growth time effect curves；
The Alternanthera philoxeroides ligroin extraction of Fig. 3 embodiments 1 suppresses MCF-7 cell IC50 curves；
The Alternanthera philoxeroides ligroin extraction of Fig. 4 embodiments 1 suppresses A549 cell IC50 curves.
Embodiment
With reference to the accompanying drawings and detailed description, the present invention is described in further detail, but the protection model of the present invention
Enclose and be not limited to the content.
Embodiment 1
The preparation method of Alternanthera philoxeroides ligroin extraction is stated described in the present embodiment, following steps are specifically included：
（1）It is 1 by Alternanthera philoxeroides and ethanol volume ratio:3 ratio immerses Alternanthera philoxeroides in ethanol solution, passes through
Ultrasonic extraction, extract to ethanol solution paler colour or it is colourless untill；
（2）Collect extract solution, use Rotary Evaporators in temperature for 47 DEG C, vacuum under conditions of 0.05Mpa to carrying
Liquid is taken to carry out vacuum distillation, concentration EtOH Sonicate extract is to without alcohol taste；
（3）Obtained EtOH Sonicate extract is collected, by the volume ratio 1 of sonicated extract and distilled water:1.5, in ultrasound
Distilled water is added in extract and is uniformly mixed so as to obtain mixed solution, is 1 by the volume ratio of mixed solution and petroleum ether:1.5 ratio exists
Add petroleum ether in mixed solution repeatedly to be extracted, until petroleum ether extraction is mutually colourless；
（4）Collect petroleum ether extract, from Rotary Evaporators temperature be 45 DEG C, vacuum be 0.04Mpa condition
Under vacuum distillation is carried out to petroleum ether extract extract is concentrated, extract absolute ethyl alcohol after concentration, which dissolves, to be obtained
Mixed solution, by mixed solution at ambient pressure, temperature be 40 DEG C under conditions of dry 12h, finally vacuum be 0.06Mpa,
Drying temperature be 40 DEG C under conditions of dry 10h after obtain drying petroleum ether extract.
The external anticancer experiment for the Alternanthera philoxeroides ligroin extraction that the present embodiment is prepared, specifically includes following step
Suddenly：
（1）Prepare following cell line, reagent and instrument：Non-small cell lung carcinoma A549, human breast carcinoma MCF-7；DMEM
Nutrient solution（GIBCO）, hyclone（GIBCO）, trypsase（GIBCO）, DMSO（Analyze pure, Amresco）, penicillin
(Sigma), streptomysin（Sigma）, MTT（Amresco）, 96 orifice plates, cell culture incubator（Thermo）, ELIASA（Tecan）,
Biological operation bench（Wujiang Lutao cleaning equipment factory）.
（2）Tumor cell culture：Basal medium 1640+10%FBS（Hyclone）+ 1% dual anti-mycillin, cultivates bar
Part is 37 DEG C, 5%CO2Concentration and saturated humidity；When cell growth to logarithmic phase, with trypsin digestion and cell, washing is dispelled
Cell, with every hole 3 × 103The individual μ l of cell 100 are inoculated in 96 orifice plates, in case MTT experiment.
（3）Cell proliferation experiment（Mtt assay）：The Alternanthera philoxeroides ligroin extraction for taking the present embodiment to prepare is dissolved in
DMSO solution, is respectively configured concentration for 100mg/ml, 50mg/ml, 25mg/ml, 12.5mg/ml, 6.25mg/ml, 3.125mg/
Ml, 1.5625mg/ml, 0.78125mg/ml, 2 times of dilutions of each concentration of totally 8 concentration；Each concentration petroleum ethers of 1 μ l are added per hole
Extract, and 100 μ l culture mediums are added, mix so that the final concentration of 500 μ g/ in every hole of Alternanthera philoxeroides ligroin extraction
ml、250μg/ml、125μg/ml、62.5μg/ml、31.25μg/ml、15.625μg/ml、7.8125μg/ml、3.90625μg/
ml；Incubator is put into continue to cultivate 24h, 48h, 72h.20 μ l MTT solution are added per hole, continue to cultivate 4h；Cultivate and extracted out after 4h
The absorbance determined under 100 μ l DMSO solutions, ELIASA per hole is added after per hole solution per hole；To add the μ l of DMSO solution 1 trainings
Foster cell is control group, and each concentration gradient sets 5 parallel groups, cell inhibitory rate is calculated respectively；Calculated according to below equation
Inhibiting rate：
Inhibiting rate=(1- (experimental group absorbance/control group absorbance)) × 100%.
Inhibitory action of the various concentrations Alternanthera philoxeroides ligroin extraction of table 1. to MCF-7 cell growths
Inhibitory action of the various concentrations Alternanthera philoxeroides ligroin extraction of table 2. to A549 cell growths
Embodiment 2
The preparation method of Alternanthera philoxeroides ligroin extraction is stated described in the present embodiment, following steps are specifically included：
（1）It is 1 by Alternanthera philoxeroides and ethanol volume ratio:5 ratio immerses Alternanthera philoxeroides in ethanol solution, passes through
Ultrasonic extraction, extract to ethanol solution paler colour or it is colourless untill；
（2）Collect extract solution, use Rotary Evaporators in temperature for 43 DEG C, vacuum under conditions of 0.06Mpa to carrying
Liquid is taken to carry out vacuum distillation, concentration EtOH Sonicate extract is to without alcohol taste；
（3）Obtained EtOH Sonicate extract is collected, by the volume ratio 1 of sonicated extract and distilled water:2, carried in ultrasound
Take addition distilled water in thing to be uniformly mixed so as to obtain mixed solution, be 1 by the volume ratio of mixed solution and petroleum ether:1 ratio is in mixing
Add petroleum ether in solution repeatedly to be extracted, until petroleum ether extraction is mutually colourless；
（4）Collect petroleum ether extract, from Rotary Evaporators temperature be 48 DEG C, vacuum be 0.05Mpa condition
Under vacuum distillation is carried out to petroleum ether extract extract is concentrated, extract absolute ethyl alcohol after concentration, which dissolves, to be obtained
Mixed solution, by mixed solution at ambient pressure, temperature be to dry 8h under conditions of 50 DEG C, finally vacuum be 0.08Mpa, it is dry
Dry temperature be 50 DEG C under conditions of dry 8h after obtain drying petroleum ether extract.
The external anticancer experiment for the Alternanthera philoxeroides ligroin extraction that the present embodiment is prepared, specific steps are with implementation
Example 1, the present embodiment the results are shown in Table 3,4.
Inhibitory action of the various concentrations Alternanthera philoxeroides ligroin extraction of table 3. to MCF-7 cell growths
Inhibitory action of the various concentrations Alternanthera philoxeroides ligroin extraction of table 4. to A549 cell growths
Embodiment 3
The preparation method of Alternanthera philoxeroides ligroin extraction is stated described in the present embodiment, following steps are specifically included：
（1）It is 1 by Alternanthera philoxeroides and ethanol volume ratio:2 ratio immerses Alternanthera philoxeroides in ethanol solution, passes through
Ultrasonic extraction, extract to ethanol solution paler colour or it is colourless untill；
（2）Collect extract solution, use Rotary Evaporators in temperature for 48 DEG C, vacuum under conditions of 0.04Mpa to carrying
Liquid is taken to carry out vacuum distillation, concentration EtOH Sonicate extract is to without alcohol taste；
（3）Obtained EtOH Sonicate extract is collected, by the volume ratio 1 of sonicated extract and distilled water:1, carried in ultrasound
Take addition distilled water in thing to be uniformly mixed so as to obtain mixed solution, be 1 by the volume ratio of mixed solution and petroleum ether:2 ratio is in mixing
Add petroleum ether in solution repeatedly to be extracted, until petroleum ether extraction is mutually colourless；
（4）Collect petroleum ether extract, from Rotary Evaporators temperature be 43 DEG C, vacuum be 0.06Mpa condition
Under vacuum distillation is carried out to petroleum ether extract extract is concentrated, extract absolute ethyl alcohol after concentration, which dissolves, to be obtained
Mixed solution, by mixed solution at ambient pressure, temperature be 45 DEG C under conditions of dry 10h, finally vacuum be 0.07Mpa,
Drying temperature be 45 DEG C under conditions of dry 12h after obtain drying petroleum ether extract.
The external anticancer experiment for the Alternanthera philoxeroides ligroin extraction that the present embodiment is prepared, specific steps are with implementation
Example 1, the present embodiment the results are shown in Table 5,6.
Inhibitory action of the various concentrations Alternanthera philoxeroides ligroin extraction of table 5. to MCF-7 cell growths
Inhibitory action of the various concentrations Alternanthera philoxeroides ligroin extraction of table 6. to A549 cell growths
The result of embodiment 2,3 before the combination of embodiment 1, obtains following Fig. 1, the result shown in 2,3,4.By table 1,
3,5 can be seen that the petroleum ether extract of Alternanthera philoxeroides is inhibited for MCF-7 breast cancer cells, and inhibitory action
It is proportional with dosage.
By table 2,4,6 it can be seen that the petroleum ether extract of Alternanthera philoxeroides has for A549 non-small cell lung cancer cells
There is inhibitory action, and inhibitory action and dosage are proportional.
It can illustrate that the petroleum ether extract of Alternanthera philoxeroides is non-to MCF-7 breast cancer cells and A549 small by Fig. 1,2
The inhibitory action of cell lung cancer cell is into dosage-Time-effect relationship, with the increase of administration time and concentration, its to this two
The inhibitory action for planting cancer cell is consequently increased, proportional.
By Fig. 3,4 the petroleum ether extract of Alternanthera philoxeroides can be drawn for MCF-7 breast cancer cells and A549
The suppression curve of non-small cell lung cancer cell, and can calculate corresponding IC50 values, respectively 30.11 μ g/ml,
9.075μg/ml 。
Claims (1)
1. utilization of the Alternanthera philoxeroides ligroin extraction in cancer therapy drug is prepared, the cancer therapy drug is used to suppress MCF-7
Breast cancer cell and A549 non-small cell lung cancer cells；
The preparation method of the Alternanthera philoxeroides ligroin extraction, specifically includes following steps：
（1）It is 1 by Alternanthera philoxeroides and ethanol volume ratio:2~1:5 ratio immerses Alternanthera philoxeroides in ethanol solution, passes through
Ultrasonic extraction, extract to ethanol solution paler colour or it is colourless untill；
（2）Extract solution is collected, EtOH Sonicate extract is concentrated to without alcohol taste using vacuum distillation；
（3）Obtained EtOH Sonicate extract is collected, by the volume ratio 1 of sonicated extract and distilled water:1~1:2, carried in ultrasound
Take addition distilled water in thing to be uniformly mixed so as to obtain mixed solution, be 1 by the volume ratio of mixed solution and petroleum ether:1~1:2 ratio exists
Add petroleum ether in mixed solution repeatedly to be extracted, until petroleum ether extraction is mutually colourless；
（4）Collect after petroleum ether extract, vacuum distillation concentration extract, extract absolute ethyl alcohol dissolves, and then carries out just
Walk after the petroleum ether extract after constant pressure and dry, preliminarily dried is dried in vacuo and obtain drying petroleum ether extract；
The Alternanthera philoxeroides is for fresh collection or by the one or more in dry rhizome, branches and leaves, fruit；
Step（2）Described in vacuum distillation select Rotary Evaporators, condition is 43 DEG C ~ 48 DEG C, vacuum is 0.04 ~
0.06MPa；
Step（4）Described in vacuum distillation select Rotary Evaporators, condition is 43 DEG C ~ 48 DEG C, vacuum is 0.04 ~
0.06MPa；
Step（4）Described in constant pressure and dry during, drying temperature be 40 DEG C ~ 50 DEG C, drying time be 8 ~ 12h, vacuum do
During dry, drying temperature is 40 DEG C ~ 50 DEG C, and drying time is 8 ~ 12h.
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Non-Patent Citations (3)
| Title |
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| 空心莲子草的化学成分及其应用;谭苹等;《公共卫生与预防医学》;20071231;第18卷(第5期);50-52 * |
| 空心莲子草的化学成分研究;罗晓茹等;《中国药学杂志》;20070831;第42卷(第15期);1138-1141 * |
| 空心莲子草的研究进展;孙永艳等;《广东农业科学》;20111231(第13期);73-77 * |
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