CN104887725B - Chenopodium ambrosiodies complete stool plants the new application of extract - Google Patents
Chenopodium ambrosiodies complete stool plants the new application of extract Download PDFInfo
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Abstract
The invention discloses the new applications of chenopodium ambrosiodies whole plants extract, belong to drug, natural medicine technical field.Application of the chenopodium ambrosiodies whole plants chloroform phase extract in preparing anti-non-small cell lung cancer drug.The preparation method of the extract is as follows：Using 95% ethyl alcohol as extractant, chenopodium ambrosiodies complete stool is extracted using ultrasonic extraction, obtains its ethyl alcohol total extract, first uses petroleum ether extraction, then extraction extraction raffinate is extracted with chloroform, concentrated extract obtains chloroform phase extract；The chloroform of chenopodium ambrosiodies whole plants provided by the present invention mutually extracts part has apparent inhibition cell Proliferation and inducing cell apoptosis effect to Non-small Cell Lung Cancer A 549.
Description
Technical field
The present invention relates to the new applications that chenopodium ambrosiodies complete stool plants extract, belong to drug, natural medicine technical field.
Background technology
All the time, malignant tumour is all to seriously threaten the principal disease of human health.In more than 20 kinds of common cancers, lung
Cancer oneself become one of maximum malignant tumour of the extent of injury.The difference for the treatment of and prognosis, lung cancer can be divided into Small Cell Lung Cancer
(small cell lung cancer, SCLC) and non-small cell lung cancer (non-small cell lung cancer,
NSCLC).NSCLC accounts for the 80%-85% of cases of lung cancer, is the main body of lung cancer, although grade malignancy is relatively low, part development is slow, should
The threat that there is transfer in kind patients with lung cancer body brings great difficulty to the determination of clinical Clinics and Practices scheme, therefore urgently
Effective drug to be developed.China's resources of medicinal plant is abundant, and the anticancer drug to develop new provides abundant resource.In recent years
The research of some come has shown that the extract of medicinal plant all shows good active anticancer in vivo and in vitro.
Chenopodium ambrosiodiesChenopodium ambrosioides L.For chenopod, annual or perennial herb, extensively
It is distributed in whole world temperate zone to torrid areas.South in China, chenopodium ambrosiodies also have extensive distribution.Research has shown that native chaste tree
Alkaloid, saponin(e and brass alcohol glycosides, tool wind-dispelling, dehumidifying, desinsection, promoting menstruation, analgesic efficacy are rich in mustard.Chenopodium ambrosiodies is in Popular Utilization
Extensively, domestic and foreign scholars have carried out years of researches to chenopodium ambrosiodies, the analysis mainly for volatile oil component and volatile oil pharmacology
The research of effect.Nearest Wu Jia is beautiful to wait volatile oil component in scholars' research chenopodium ambrosiodies to grow tool to human breast carcinoma cell lines MCF-7
There is the apoptosis of apparent inhibiting effect and inducing cell.But there is presently no about chenopodium ambrosioides extract anti-lung cancer activity
Research report.
Invention content
The object of the present invention is to provide a kind of new application of chenopodium ambrosiodies whole plants chloroform extract, which is native chaste tree
Application of the mustard whole plants extract in preparing anti-non-small cell lung cancer drug.
Another object of the present invention is to provide the preparation methods of the chenopodium ambrosiodies whole plants chloroform phase extract, specifically
Include the following steps：
（1）Soaked overnight is carried out to chenopodium ambrosiodies branches and leaves using 95% ethyl alcohol, then ultrasonic extraction（At 50 ~ 80 DEG C, power is
Under the conditions of 200W）At least 3 times, 0.5 ~ 2h is extracted every time, and decompression, which filters, obtains ethanol extract, then repeatedly uses the second of same volume
Alcohol impregnates and repeats the extraction flow of first time, until ethanol extract is colourless, finally merges all ethanol extracts.
（2）The ethanol extract of chenopodium ambrosiodies whole plants is evaporated under reduced pressure using Rotary Evaporators, ethyl alcohol is obtained and carries
Object medicinal extract is taken, then it is dissolved with distilled water.
（3）The aqueous solution of chenopodium ambrosiodies branches and leaves ethanol extract is repeatedly extracted using petroleum ether organic solvent, until
Until extraction phase is colourless, then extraction extraction raffinate with chloroform repeatedly extract up to colourless, merges multiple extract liquor and obtain chloroform phase
Extract.
Beneficial effects of the present invention：
（1）Present invention application of the exploratory development chenopodium ambrosiodies chloroform phase extract on anti-non-small cell lung cancer for the first time.
（2）Experiment proves that chenopodium ambrosiodies whole plants chloroform phase extract of the invention is to Non-small cell lung carcinoma cell
The proliferation of A549 cells has a degree of inhibiting effect, with the increase of extract activity and prolonging for action time
It is long, apparent dosage-time-dependent effect is showed to the inhibiting effect of A549 cell growths；Hoechst is dyed and streaming is thin
Born of the same parents' instrument testing result shows that chenopodium ambrosiodies chloroform phase extract can cause chromatin bunching, phase cell cycle G1 to block and induce early
The cell proportion of phase apoptosis increases, and apparent dose-dependence is presented；Therefore the present invention is for preparing anti-non-small cell
Lung-cancer medicament is of great significance.
Description of the drawings
Fig. 1 is growth inhibition column diagram of the chenopodium ambrosiodies whole plants chloroform phase phase extract to A549 cells；
Fig. 2 is chenopodium ambrosiodies whole plants chloroform extract to A549 cell clonal formation dose curve figures；
Fig. 3 is chenopodium ambrosiodies whole plants chloroform phase extract to A549 nuclei dyeing chromatic graphs；
Fig. 4 is flow cytomery chloroform extract to A549 Cell cycle influences figures；
Fig. 5 is flow cytomery chloroform extract to A549 early apoptosis of cells action diagrams.
Specific implementation mode
Invention is further described in detail with reference to the accompanying drawings and detailed description, but protection scope of the present invention
It is not limited to the content.
Embodiment 1
The preparation of chenopodium ambrosiodies whole plants chloroform phase extract, specifically includes following steps：
（1）It dries in the shade to the complete stool chenopodium ambrosiodies branches and leaves collected, obtains dry chenopodium ambrosiodies whole plants
213.46g mixes soaked overnight with 95% ethyl alcohol of concentration, then ultrasonic extraction 3 times, and the temperature of ultrasonic extraction is 50 DEG C, and power is
200W extracts 0.5h every time, filtrate 1 then is obtained by filtration in ultrasonic extraction liquid, then with 95% second of concentration identical with initial proportion
Alcohol impregnates filter residue, constantly repeats above-mentioned identical extraction flow, obtains filtrate 2-n, is until 95% ethanol extract of concentration is colourless
Only, finally merge all filtrate, obtain total 95% ethanol extract of concentration.
（2）It is evaporated under reduced pressure using Rotary Evaporators concentration 95% ethanol extract total to chenopodium ambrosiodies, obtains concentration
95% ethanol extract medicinal extract, then dissolves it with distilled water.
（3）The aqueous solution of chenopodium ambrosiodies branches and leaves ethanol extract is repeatedly extracted using petroleum ether organic solvent, until
Until extraction phase is colourless, then extraction extraction raffinate with chloroform organic solvent repeatedly extract up to colourless, utilizes Rotary Evaporators
Above-mentioned chloroform extraction phase is evaporated under reduced pressure, the medicinal extract of chenopodium ambrosiodies whole plants chloroform recovery part is obtained.
Embodiment 2
（1）It dries in the shade to the complete stool chenopodium ambrosiodies branches and leaves collected, obtains dry chenopodium ambrosiodies whole plants
213.46g mixes soaked overnight with 95% ethyl alcohol of concentration, then ultrasonic extraction 4 times, and the temperature of ultrasonic extraction is 60 DEG C, and power is
200W extracts 1h every time, filtrate 1 then is obtained by filtration in ultrasonic extraction liquid, then with 95% ethyl alcohol of concentration identical with initial proportion
Filter residue is impregnated, above-mentioned identical extraction flow is constantly repeated, obtains filtrate 2-n, be until 95% ethanol extract of concentration is colourless
Only, finally merge all filtrate, obtain total 95% ethanol extract of concentration.
（2）It is evaporated under reduced pressure using Rotary Evaporators concentration 95% ethanol extract total to chenopodium ambrosiodies, obtains concentration
95% ethanol extract medicinal extract, then dissolves it with distilled water.
（3）The aqueous solution of chenopodium ambrosiodies branches and leaves ethanol extract is repeatedly extracted using petroleum ether organic solvent, until
Until extraction phase is colourless, then extraction extraction raffinate with chloroform organic solvent repeatedly extract up to colourless, utilizes Rotary Evaporators
Above-mentioned chloroform extraction phase is evaporated under reduced pressure, the medicinal extract of chenopodium ambrosiodies whole plants chloroform recovery part is obtained.
Embodiment 3
（1）It dries in the shade to the complete stool chenopodium ambrosiodies branches and leaves collected, obtains dry chenopodium ambrosiodies whole plants
213.46g mixes soaked overnight with 95% ethyl alcohol of concentration, then ultrasonic extraction 5 times, and the temperature of ultrasonic extraction is 80 DEG C, and power is
200W extracts 1.8 h, filtrate 1 then is obtained by filtration in ultrasonic extraction liquid, then with concentration identical with initial proportion 95% every time
Ethyl alcohol impregnates filter residue, constantly repeats above-mentioned identical extraction flow, obtains filtrate 2-n, until 95% ethanol extract of concentration is colourless
Until, finally merge all filtrate, obtains total 95% ethanol extract of concentration.
（2）It is evaporated under reduced pressure using Rotary Evaporators concentration 95% ethanol extract total to chenopodium ambrosiodies, obtains concentration
95% ethanol extract medicinal extract, then dissolves it with distilled water.
（3）The aqueous solution of chenopodium ambrosiodies branches and leaves ethanol extract is repeatedly extracted using petroleum ether organic solvent, until
Until extraction phase is colourless, then extraction extraction raffinate with chloroform organic solvent repeatedly extract up to colourless, utilizes Rotary Evaporators
Above-mentioned chloroform extraction phase is evaporated under reduced pressure, the medicinal extract of chenopodium ambrosiodies whole plants chloroform recovery part is obtained.
Application of the chenopodium ambrosiodies whole plants chloroform extract obtained in embodiment 1 ~ 3 in inhibiting growth of cancer cells
By non-small cell lung cancer cell A549 cultures in the mixed liquor containing 10% fetal calf serum, 1% penicillin and streptomysin
In RPMI-1640 complete mediums, 37 DEG C are placed in, 5%CO2Incubator in, after cell enters exponential phase, be added
0.25% EDTA- trypsin solution vitellophags, are then adjusted to a concentration of 5 × 10 with RPMI-1640 complete mediums4
The cell suspension of a/ml；It is inoculated in again in 96 well culture plates, per 100 μ l of hole, in 37 DEG C, 5%CO2Incubator in train
Supporting rs for 24 hours keeps cell adherent.
（1）Chenopodium ambrosiodies whole plants chloroform phase extract obtained in embodiment 1,2,3 is dissolved in DMSO, is made dense
Degree is that the screening mother liquor of 100mg/ml is spare.
（2）Doubling dilution is carried out using DMSO, respectively obtains chloroform phase extract final concentration of 500 μ g/ml, 250 μ g
Examination is respectively set in/ml, 125 μ g/ml, 62.5 μ g/ml, 31.25 μ g/ml, the DMSO solution of 15.63 μ g/ml, 7.81/ml
It tests group and control group is tested.
Test group：Chenopodium ambrosiodies chloroform phase extract is added in the A549 cell culture wells of 96 orifice plates, 1 μ is added per hole
L, then 100 μ lRPMI-1640 complete mediums are added into each culture hole；Different extract activities, native chaste tree are set
Mustard chloroform phase extract solution is according to final concentration of 500 μ g/ml, 250 μ g/ml, 125 μ g/ml, 62.5 μ g/ml, and 31.25
μ g/ml, 15.63 μ g/ml and 7.81 μ g/ml each handle 5 repeating holes of setting.
Control group：DMSO is added in the culture hole of non-small cell lung cancer A549,1 μ l are added per hole, then again to each training
It supports in hole and adds 100 μ lRPMI-1640 complete mediums, 5 DMSO negative control holes are set altogether.
Above-mentioned culture plate is placed in 37 DEG C, 5%CO2Incubator in continue cultivate 72hrs.
The tetramethyl azo azoles salt of a concentration of 5mg/ml of 20 μ l Fresh is added after 72hrs to every hole（MTT）Solution,
And at 37 DEG C, 5%CO2Under conditions of continue cultivate 4hrs after carefully inhale abandon supernatant.The DMSO of 100 μ l is added per hole, shakes at room temperature
10min is swung, light absorption value of each hole at 490nm is measured on multi-function microplate reader（OD values）, growth of cancer cells is calculated by formula
Inhibiting rate：Cancer cell multiplication inhibiting rate=（The average OD values that the average OD values that control wells measure-dosing group measures）/ control
Average OD value × 100% that hole measures.
Analyzing processing is carried out to result using 5 softwares of GraphPad Prism, chenopodium ambrosiodies whole plants chloroform is obtained and mutually carries
Take object to the half-inhibition concentration after non-small cell lung cancer A549 effects 72hrs（IC50）, as a result as shown in table 1 below：
Table 1
The chloroform phase extract of chenopodium ambrosiodies whole plants is grown after acting on rs, 48hrs, 72hrs for 24 hours respectively to A549 cells
The docs-effect column diagram of inhibition is as shown in Figure 1.As a result the increase with extract-treated cell concentration and action time are shown
Extension, the growth inhibition of A549 cells is significantly increased, apparent dosage-time-dependent effect is presented.
Cell clonal formation is tested：
After A549 cells enter exponential phase, 0.25% EDTA- trypsin solution vitellophags are added.Use RPMI
Cell is diluted to the cell suspension that density is 200/ml by 1640 culture medium, is added in 6 porocyte culture plates, is added per hole
1ml tumor cell suspensions.It waits for that the adherent rs for 24 hours of cell is added and final concentration of the 7.81,15.63 of 10 μ l is respectively added into each hole,
31.25,62.5,125 μ g/ml and 250 μ g/ml chloroform phase phase extracts, the DMSO of 10 μ l is added in control group, then is mended to each hole
Add the RPMI-1640 culture mediums of 1ml；Culture medium is abandoned after continuing culture 12 days, 4% poly methanol is fixed, and violet staining is micro-
Under the microscope, the number of cell clones that cell number is more than 50 is calculated；The results are shown in Figure 2, with the increase of extract concentrations, is formed
Cell clone number significantly reduce, apparent dose dependent is presented to the toxicity of cell in extract.
Hoechst33342 dye tests：
After A549 cells enter exponential phase, 0.25% EDTA- trypsin solution vitellophags are added.Use RPMI
It is 1 Χ 10 that cell is diluted to density by 1640 culture medium5The cell suspension of a/ml is added in 24 porocyte culture plates, adds per hole
Enter 1ml tumor cell suspensions.It waits for that the adherent rs for 24 hours of cell is added the chenopodium ambrosiodies chloroform of 10 μ l is respectively added into each hole mutually to extract
Object（Final concentration of 74.56 μ g/ml）, the DMSO of 10 μ l is added in control group, then the RPMI1640 cultures of 1ml are added to each hole
Base continues culture rs for 24 hours.Culture medium is discarded, PBS is washed 1 time, and 4% paraformaldehyde fixes 15-20min, and PBS is washed 2 times,
Hoechst33342 dyeing is protected from light dyeing 30min at room temperature, and PBS is washed 2 times, with the observation of inverted fluorescence microscope blue-fluorescence, bat
According to.As a result if figure is in chenopodium ambrosiodies chloroform phase extract I C50Concentration（74.56μg/ml）When as shown in Figure 3B with cell controls group（Figure
3A）It compares, nuclear chromatin shrinkage occurs in the cell of dosing object, the segment that crescent is either broken occurs, and brightness increases, hence it is evident that
Early apoptosis of cells cytology phenotype
Cell cycle is detected：
After A549 cells enter exponential phase, 0.25% EDTA- trypsin solution vitellophags are added.Use RPMI
It is 5 Χ 10 that cell is diluted to density by 1640 culture medium5The cell suspension of a/ml is added in 6 porocyte culture plates, adds per hole
Enter 1ml tumor cell suspensions.Continue to cultivate the chenopodium ambrosiodies chloroform phase extract that 10 μ l are respectively added into hole by rs for 24 hours after inoculation（Eventually
A concentration of 74.56 μ g/ml）, the DMSO of 10 μ l is added in control group, then the RPMI-1640 culture mediums of 1ml are added to each hole, after
It is continuous to cultivate rs for 24 hours.With trypsin digestion cell, 1000g centrifuges 3-5 minutes sedimentation cells.It is pre- that 1ml ice baths are added in careful absorption supernatant
Cell is resuspended in cold PBS, and centrifugation cell, carefully absorbs supernatant again.The PBS of 1ml ice baths precooling is added again, is resuspended
Cell.1000g centrifuges 3-5 minute sedimentation cells, 4 DEG C of fixations rs for 24 hours after the 95% ethyl alcohol resuspension cell mixing of 75% ice bath precooling.
Fixer is washed away, propidium iodide stain is protected from light at room temperature is incubated 30min, flow cytomery, result Flowjo.7.6
Software is analyzed.As a result the chenopodium ambrosiodies whole plants chloroform phase extract as shown in Fig. 4 is with IC50Concentration processing cell after（Figure
4B）, with cellular control unit（Fig. 4 A）It is significantly arrested in the G1 phases compared to the cell cycle, as a result shows cell-cycle arrest in the G1 phases
(the VS blank controls of X ± SD, n=3, * P<0.05, * * P<0.01, * * * P<0.001).
Apoptosis detects：
After A549 cells enter exponential phase, 0.25% EDTA- trypsin solution vitellophags are added.Use RPMI
It is 5 Χ 10 that cell is diluted to density by 1640 culture medium5The cell suspension of a/ml is added in 6 porocyte culture plates, adds per hole
Enter 1ml tumor cell suspensions.Continuing culture after inoculation, 10 μ l chenopodium ambrosiodies chloroform phase extracts are added in rs for 24 hours（It is final concentration of
74.56μg/ml）, the DMSO of 10 μ l is added in control group, then the RPMI-1640 culture mediums of 1ml are added to each hole, continues to cultivate
24hrs.The PBS being pre-chilled with 4 DEG C washes cell twice, and cell is resuspended again with 250 μ l combination buffers, 5 μ l Annexin are added
The ofpropidium iodide solution of 20 μ g/ml of V/FITC and 10 μ l is protected from light after mixing in room temperature and is incubated 15min.It is examined with flow cytometer
It surveys, result is analyzed with Flowjo.7.6 softwares.The results are shown in Figure 5 chenopodium ambrosiodies whole plants chloroform phase extract with
IC50Concentration processing cell after（Fig. 5 B）, with cellular control unit（Fig. 5 A）Compared to the apoptosis that extract can significantly cause cell
Effect.As a result：Extract inducing cell early apoptosis (the VS blank controls of X ± SD, n=3, * P<0.05, * * P<0.01, * * * P<
0.001)。
Claims (3)
1. application of the chenopodium ambrosiodies whole plants extract in preparing anti-non-small cell lung cancer drug；
The preparation method of the chenopodium ambrosiodies whole plants extract specifically includes following steps：
（1）Soaked overnight is carried out to chenopodium ambrosiodies branches and leaves using ethyl alcohol, 0.5 ~ 2h is extracted in then ultrasonic extraction at least 3 times every time,
Decompression, which filters, obtains ethanol extract, then is repeatedly impregnated with the ethyl alcohol of same volume and repeated the extraction flow of first time, Zhi Daoyi
Until alcohol extract is colourless, finally merge all ethanol extracts；
（2）The ethanol extract of chenopodium ambrosiodies branches and leaves is evaporated under reduced pressure using Rotary Evaporators, obtains ethanol extract medicinal extract,
Then it is dissolved with distilled water；
（3）The aqueous solution of chenopodium ambrosiodies branches and leaves ethanol extract is repeatedly extracted using petroleum ether organic solvent, until extraction
Until mutually colourless, then extraction extraction raffinate with chloroform repeatedly extract up to colourless, merges multiple extract liquor and obtain chloroform and mutually extract
Object.
2. chenopodium ambrosiodies whole plants extract according to claim 1 answering in preparing anti-non-small cell lung cancer drug
With, it is characterised in that：The temperature of the ultrasonic extraction is 50 ~ 80 DEG C, power 200W.
3. chenopodium ambrosiodies whole plants extract according to claim 1 answering in preparing anti-non-small cell lung cancer drug
With, it is characterised in that：The concentration of volume percent of the ethyl alcohol is 95%.
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| CN101639464A (en) * | 2008-07-30 | 2010-02-03 | 天津天士力制药股份有限公司 | Method for analyzing ascaridole content in blood plasma and application thereof in pharmacokinetics |
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| CN101639464A (en) * | 2008-07-30 | 2010-02-03 | 天津天士力制药股份有限公司 | Method for analyzing ascaridole content in blood plasma and application thereof in pharmacokinetics |
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| 倍半萜内酯化合物药理作用;徐静等;《中国热带医学》;20070430;第7卷(第04期);第623-624页 * |
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