CN105125748A - Preparation method and application of congea chinensis extract - Google Patents
Preparation method and application of congea chinensis extract Download PDFInfo
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Abstract
The invention discloses a preparation method for an active extract extracted from a Yunnan featured plant, namely, congea chinensis. The extract is obtained through organic solvent digestion, extraction and open chromatograph group separation, has remarkable antitumor activity, and can targetedly activate tumor inhibiting gene p53; besides, the extract has no obvious toxicity on normal cells, which shows that the novel active extract component of the Yunnan featured plant, namely, congea chinensis has high application potential in preparation of low-toxicity, efficient, antitumor and personalized treating medicine.
Description
Technical field
The invention belongs to field of medicaments, be specifically related to a kind of Yunnan Special plant China floss bud rattan (
congeachinensis) extract preparation method and preparing the application in selectivity antineoplaston medicine.
Background technology
Tumor is the fatal disease of a class serious threat human health, and its pathogenesis is illustrated not yet completely, and therapeutic effect is also unsatisfactory.Current routine is used for most of medicine of oncotherapy, is all to design for the regulation and control of DNA replication dna in proliferation process or cytoskeleton.These medicines are while killing and wounding the tumor cell of fast breeding, required normal proliferating cells (as bone marrow stem cell etc.) is maintained to bodily fuctions and also has very large toxicity, cause connecing subject patient's body normal function to be badly damaged, resistivity for tumor declines on the contrary, is difficult to get good therapeutic effect.Therefore, utilize molecular targeted triage techniques, for tumor cell specific gene mutation product, screening can suppress the micromolecular compound of these tomour specific mutating molecules activity or expression, thus exploitation forms low toxicity, efficiently targeting personalized treatment medicine, has become the recent tendency of anti-tumor medicine research and development.Confirmed that Mutation p53 is the target spot of antitumor drug, at present, the antitumor drug research being shot design for Mutation p53 enters clinical trial.Therefore, exploitation has low toxicity, efficiently the targeting personalized treatment medicine of independent intellectual property right, also becomes the important directions of domestic tumor transformation medical investigator.
In cell, the content of p53 albumen is very low under normal circumstances, and Mutation p53 usually has the expression of higher level in tumor cell, thus becomes and be different from a Normocellular specificity antineoplastic target spot.At present, Therapeutic Method for Mutation p53 mainly relies in tumor cell the expression reactivating wild type p53 or way Mutation p53 being reverted to wild type p53, be exactly the treatment that the targeting Mutation p53 strategy that makes it degrade carrys out targeting Mutation p53 in addition, mainly polypeptide, micromolecular compound etc., these compounds are all abroad find according to the structural design of p53, and mainly from Yunnan Province, distinctive plant resources separation and Extraction finds to have the compound that can activate wild type p53 in the present invention.Detection method p53 promoter being connected GFP reporter gene filters out the compound that can act on p53 promoter intuitively by the change detecting fluorescence, particularly this system is applied to the screening report of plant compound seldom.
Yunnan Province is one of hot zones of world's bio-diversity, and various biological species accounts for more than 50% of the whole nation, and the research and development for natural drug provides important material base.Simultaneously 26, Yunnan nationality with the long-term struggle of disease in have accumulated abundant national folk medication experience, for Yunnan researches on natural drugs provides valuable traditional knowledge, medicine background information and data, also for the natural medicinal plant of our R and D targeting therapy on tumor provides abundant source.
China floss bud rattan (
congeachinensis), originate from screen limit, Yunnan and Xishuangbanna, often grow on the cheuch woods limit of height above sea level 700-1450 rice, mainly as ornamental plant.Up to now, the report having anti-tumor activity about magnificent floss bud rattan extract active component is had no.
Summary of the invention
The present invention, in order to expand the frontier of Yunnan Special plant research, makes full use of China's plant resources, develops its novelty teabag, provides a kind of preparation method of magnificent floss bud rattan extract; Realized by following steps:
(1) pulverizing and lixiviate
After plant China floss bud rattan is pulverized, with pharmaceutical grade methanol room temperature lixiviate 4-5 days, sucking filtration, filtrate concentrates, and obtains methanol extract study, by ethyl acetate and water extract and separate, obtains water layer extractum;
(2) abstraction and purification
By water layer extractum through non-polar macroporous resin pillar layer separation, solvent system is ethanol-water mixture, collects and merges ethanol: water volume ratio is the elution fraction of 8:2, concentrated, obtain magnificent floss bud rattan crude extract Fr-3 component, by crude extract Fr-3 component water dissolution, methanol extraction, filter, and precipitate 3 times by methanol wash, precipitate through ODS pillar layer separation, solvent system is methanol-water mixture, collect and merge methanol: water volume ratio is the elution fraction of 8:2, concentrated, obtain magnificent floss bud rattan extract.
This extract is in white, tasteless, soluble in water, be dissolved in dimethyl sulfoxide, be slightly soluble in ethanol and methanol, when analyzing with TLC, with chloroform: methanol=1:1 is developing solvent, be that on 5cm forward Silica plate, its Rf value is about 0.5-0.6 at height, this sample has ultraviolet absorption peak under 210nm wavelength, and the content in magnificent floss bud rattan is 0.05 ‰.
Another object of the present invention is applied to by said extracted thing to prepare antitumor personalized treatment medicine, prepares the medicine to not normal cells.
Extract of the present invention to the embryo fibroblast (mouseembryonicfibroblast, MEF) of wild-type mice without obvious cytotoxicity.
Beneficial effect of the present invention is:
(1) organic solvent lixiviate, solvent distribute, opening chromatographic column is separated and obtains, and preparation method has the advantages such as simple and quick;
(2) the present invention utilizes a kind of Yunnan of transgenic mouse cell recombinant target gene constructed p53 promoter-GFP molecule report carrier targeting Screening and Identification Special plant China floss bud rattan extract active component to the activation situation of p53 gene; Data shows, this component can obviously activate GFP fluorescence, has potential anti-tumor activity;
(3) MEF cell in contrast, the application of this normal control cells in drug screening system can get rid of the cell killing effect caused because of cytotoxic effect, screening kills and wounds tumor cell is selective, and does not have virose natural active matter to normal cell;
(4) cellular pharmacology experiment proves, magnificent floss bud rattan extract can kill most tumor cell, and less to MEF cytotoxicity, illustrates that its antitumor action has good selectivity.
The present invention has opened up the new medicinal usage of magnificent floss bud rattan extract active component, and the personalized treatment for tumor provides new low toxicity, efficiently medicine.
Accompanying drawing explanation
Fig. 1 is that the Differential Interference Contrast figure DIC(A of negative control 1%DMSO in the present invention schemes) and GFP fluorogram (B figure);
Fig. 2 adds embodiment 1 magnificent floss bud rattan extract active component p53 after process in 72 hours
-/-the Differential Interference Contrast figure DIC(A of-p53-GFPMEF cell schemes) and GFP fluorogram (B figure);
Fig. 3 adds embodiment 1 magnificent floss bud rattan extract active component, after process in 72 hours, and p53 protein expression showed increased in A549 cell;
Fig. 4 is that the Differential Interference Contrast figure DIC(A of negative control 1%DMSO in the present invention schemes) and GFP fluorogram (B figure);
Fig. 5 is that the Differential Interference Contrast figure DIC(A of the embodiment of the present invention 5 Sinensis bud rattan extract active component schemes) and GFP fluorogram (B figure);
Fig. 6 is the expression of results that in the present invention, embodiment 5 magnificent floss bud rattan extract active component obviously can increase p53 albumen in A549.
Detailed description of the invention
Again the present invention is described in further detail below by the embodiment and accompanying drawing, subordinate list of being prepared by this active component to example; but protection scope of the present invention is not limited to described content; in embodiment, method all adopts conventional method if no special instructions; use reagent if no special instructions, the reagent being conventional commercial reagent or adopting conventional method to configure.
embodiment 1:
the preparation of China's floss bud rattan extract active component
In December, 2014 is from collection Yunnan, Miao Autonomous County of Pingbian, Yunnan Province China floss bud rattan aerial parts sample 1kg, plant species name is identified by Yunnan Province Agriculture Academy of Science Medicine Plant Institute researcher, and crude drug specimen deposits in Yunnan Province Agriculture Academy of Science Medicine Plant Institute.
From plant China floss bud rattan, obtain the preparation method of the active component with remarkable anti-tumor activity, concrete steps are:
(1) pulverizing and lixiviate
After 1kg Medicinal Plants in Yunnan China floss bud rattan aerial parts is shone dry grinding, with lixiviate under 10L methanol (pharmaceutical grade) room temperature 5 days (shaking once in a while), after sucking filtration filtrate is concentrated, ethyl acetate (400ml) and water (400ml) is used to extract three times again, concentrated after three water layer extracts are merged, obtain water layer extractum for subsequent use;
(2) abstraction and purification
Water layer extractum is separated (following solvent system all by volume, ethanol: water=0:100 through non-polar macroporous resin D101 post; 40:60; 80:20; 100:0), the elution fraction of required active component main Yi Chun ﹕ water=80 ﹕ 20, merges this position concentrated, obtains active component and slightly extract Fr-3(3.2g).Fr-3 component use water (20mL) dissolved, methanol (50mL) precipitates, and filters, and precipitates 3 times by methanol wash, must precipitate Fr-3c(0.137g).Fr-3c is through ODS pillar layer separation, and solvent system is methanol: water (0:100,50:50,80:20,100:0), collects and merges methanol: the elution fraction of water=80:20, concentrated, obtains magnificent floss bud rattan activity extract (50mg).
Through the active component that said method is prepared into, in white, tasteless, soluble in water, be dissolved in dimethyl sulfoxide, be slightly soluble in ethanol and methanol, when analyzing with TLC, with chloroform: methanol=1:1 is developing solvent, be on 5cm forward Silica plate at height, its Rf value is about 0.5-0.6, and this sample has significant ultraviolet absorption peak under 210nm wavelength, and the content in magnificent floss bud rattan is 0.05 ‰.
embodiment 2: embodiment 1 magnificent floss bud rattan extract active component activates p53
-/-
gFP fluorescence experiments in-p53-GFPMEF cell, specific experiment operation is as follows:
(1) structure of p53-GFP-report carrier, concrete grammar is as follows:
1) adopt BgLII and NotI double digestion to be cut from pAcGFP1-N1 plasmid (buying in Clontech) by green fluorescent protein GFP reporter gene, replace pGL4.82(and buy in Promega) middle Luciferase reporter gene, obtained pGL4-GFP carrier;
2) p53 gene promoter area primer (5'TGGCTCGAGGTCTTTACAGAGAGTG3' is designed, 5'CGAGATCTCGGAGAAGCGTGACA3') increase promoter sequence be connected into pGL4-GFP carrier, so just obtains report carrier-----the p53-GFP-report carrier merged with p53 gene promoter and GFP;
(2) proceeded in l cell (MEF) by the report carrier that p53 gene promoter and GFP merge, concrete grammar is as follows:
1) extract plasmid by the large extraction reagent kit of TIANGENBIOTECH plasmid and obtain pGL4.82-p53promoter-GFP plasmid, and measure its concentration;
2) p53 is cultivated
-/
-mice MEF cell, cultivation and DMEM culture medium and 10%FBS(hyclone under normal condition), by every ware 2 × 10 before transfection
6individual (10cm) goes down to posterity for one day, within second day, uses lipofectamine Lipofectamine when cell confluency rate arrives 90%
tM2000(buys in Invitrogen) carry out transfection, particular content is as follows:
A, 24 μ gpGL4.82-p53promoter-GFP plasmids are joined 3mL without in the DMEM culture medium of hyclone, obtain A mixed liquor;
B, 60 μ LLipofectamine2000 are added the DMEM culture medium of 3mL serum-free, ambient temperatare puts 20 minutes, obtains B mixed liquor;
C, to be mixed with B liquid by A liquid, be made into 24 μ g plasmids: 60 μ LLipofectamine2000 mixed liquors, after soft mixing, ambient temperatare puts 20 minutes, obtains C mixed liquor;
D, suck p53
-/
-mice MEF Tissue Culture Dish in culture medium and with 1 × PBS buffer solution for cleaning twice, add 3mL plasma-free DMEM medium, and slowly add C mixed liquor mix;
E, to be placed in by cell after 37 DEG C of cell culture incubators cultivate 5 hours, the full culture medium be replaced by containing 10% serum is cultivated;
F, add puromycin (puromycin, 2 μ g/mL) screening, contrast is set and (does not carry out the p53 of transfection
-/
-mice MEF cell, when untransfected, resistance be there is no to puromycin, adds after puromycin dead, with puromycin-resistant label on plasmid), can stable transfected cells be obtained after screening 2 weeks;
(3) method of active component is screened: add DMEM (10%FBS) subculture in vitro separately at stable transfection in p53-GFP cell and cultivate.Get two kinds of each 2 wares of cell, suck supernatant, add 5mLPBS(phosphate buffer) clean once, after trypsinization, add 4mL culture medium, collecting cell enters centrifuge tube 1200rpm, and 5min is centrifugal, abandons supernatant, add the DMEM culture medium of 7mL serum-free, resuspension, gets 100 μ L and carries out cell counting, by cell number 1 × 10
5every hole kind enters 6 orifice plate incubated overnight, reaches about the 70% magnificent floss bud rattan extract active component adding antitumoral compounds 20 μ g/ml and processes, the change in fluorescence of observation of cell after 72 hours in cell confluency.
experimental result:see Fig. 1,2, under the concentration of 20 μ g/ml, after 72 hours, magnificent floss bud rattan extract active component obviously can activate p53
-/-the expression of GFP fluorescence in-p53-GFPMEF cell.In Fig. 1, A, B are respectively Differential Interference Contrast figure DIC and the GFP fluorogram of negative control 1%DMSO; In Fig. 2, A, B are respectively Differential Interference Contrast figure DIC and the GFP fluorogram of drug treating 20 μ g/ml China floss bud rattan extract active component.
embodiment 3: the magnificent floss bud rattan extract active component of embodiment 1 has obvious lethal effect to tumor cell, more weak to normal cell (WT-MEF cell) lethal effect
SRB(SulforhodamineB, SRB) be a kind of pink colour anion binding dyestuff; It can specifically in living cells the basic amino acid of biomacromolecule be combined, 540nm wavelength place enzyme connection detector measure its light absorption value, the size of light absorption value then represents the relative populations of living cells.SRB dyeing is not easy variable color compared with MTT, and stability is better, also can preserve the long period, so light absorption value can not be much affected after cell is fixing and after dissolving with Tris-base.In addition, when measuring the medicine of band pigment because the process containing eluting, can the upper impact getting rid of pigment largely.This experiment adopts SRB to detect magnificent floss bud rattan extract active component to the impact of A549 cell, HT29 cell and WT-MEF ability of cell proliferation.
Experiment main material: A549 cell, HT29 cell, WT-MEF cell, SRB dyestuff
Experimental implementation: collect logarithmic (log) phase cell, adjustment concentration of cell suspension, be inoculated on 96 orifice plates by A549 cell and HT29 cell, 2500/hole, WT-MEF inoculates 5000/hole, and every hole solution final volume 200 μ L(edge hole culture medium is filled), 5%CO
237 DEG C of cultivations, the active component of the present invention of Concentraton gradient is added respectively after 12h, 6 concentration are arranged for activity of tumor cells component, be respectively 0 μ g/ml, 0.5 μ g/ml, 1 μ g/ml, 2 μ g/ml, 4 μ g/ml, 8 μ g/ml, the Concentraton gradient arranged for WT-MEF cytoactive component is 0 μ g/ml, 5 μ g/ml, 10 μ g/ml, 20 μ g/ml, 40 μ g/ml, 80 μ g/ml, if 3 parallel holes, and 5%CO
2, cultivate after 72 hours for 37 DEG C, every hole adds the trichloroacetic acid of 100 μ l.2h is fixed in 4 DEG C of chromatography cabinets; Blot the liquid in orifice plate, back-off in absorbent paper, dry 1h; Every hole adds the SRB dyestuff of 100 μ l, reaction 30min.Glacial acetic acid with 1% washes 4 times, is upside down in dried overnight in absorbent paper.Add the Tris-base of the 10mM of 200 μ l, on shaking table, low speed concussion 10min, makes it fully dissolve.Enzyme connection detector detects the light absorption value under 490nm wavelength.
Experimental result (table 1): magnificent floss bud rattan extract active component has significant growth inhibitory activity to tumor cell, A549 cell IC
50be 3.51 ± 0.26 μ g/ml, HT29 cell IC
50be 3.80 ± 0.05 μ g/ml, more weak to the suppression of WT-MEF cell, IC
50be 14.31 ± 0.34 μ g/ml, illustrate that this active component all has potential antitumor action to the tumor cell that p53 is wild type and saltant type, more weak to normal cell (WT-MEF cell) lethal effect.
Table 1: the selectivity anti-tumor activity result of magnificent floss bud rattan extract active component
。
embodiment 4: embodiment 1 magnificent floss bud rattan extract active component increases the expression of p53 albumen in A549 cell
experiment main material: A549 cell
experimental implementation:
The process of variable concentrations (2.5 μ g/ml, 5 μ g/ml, 10 μ g/ml) magnificent floss bud rattan extract active component cultivate the A549 cell after 72h with 0.25% trypsinization, the centrifugal 5min of 1500rpm/min, washes 3 times with PBS, adds cell pyrolysis liquid.By 10%SDS polyacrylamide gel, electrophoresis is carried out to cell sample, after electrophoresis terminates by protein delivery to PVDF membrane (pvdf membrane, MILLIPORE, the U.S.), close 1h with the TBS containing 5% defatted milk powder, add primary antibodie (GAPDH purchased from American MILLIPORE biotech company, p53 purchased from American CST biotech company) 4 DEG C spend the night, rinse 3 times with TBS, add two anti-(GE of horseradish peroxidase-labeled, the U.S.), room temperature 2h.Detect with chemiluminescence detection system (Beijing 61 Bioisystech Co., Ltd, China).
Experimental result: compared with DMSO matched group, magnificent floss bud rattan extract active component obviously can increase the expression of p53 albumen in A549, and has certain concentration dependent (Fig. 3).
embodiment 5: the preparation of magnificent floss bud rattan extract active component
Raw material sources are with embodiment 1, and concrete steps are:
(1) pulverizing and lixiviate
After 1kg Medicinal Plants in Yunnan China floss bud rattan aerial parts is shone dry grinding, with lixiviate under 10L methanol (pharmaceutical grade) room temperature 4 days (shaking once in a while), after sucking filtration filtrate is concentrated, ethyl acetate (800ml) and water (500ml) is used to extract three times again, concentrated after three water layer extracts are merged, obtain water layer extractum for subsequent use;
(2) abstraction and purification
Water layer extractum is separated (following solvent system all by volume, ethanol: water=0:100 through non-polar macroporous resin AB-8 post; 40:60; 80:20; 100:0), the elution fraction of required active component main Yi Chun ﹕ water=80 ﹕ 20, merges this position concentrated, obtains active component and slightly extract Fr-3(3.1g).Fr-3 component use water (20mL) dissolved, methanol (50mL) precipitates, and filters, and precipitates 3 times by methanol wash, must precipitate Fr-3c(0.128g).Fr-3c is through ODS pillar layer separation, and solvent system is methanol: water (0:100,50:50,80:20,100:0), collects and merges methanol: the elution fraction of water=80:20, concentrated, obtains magnificent floss bud rattan activity extract (47mg).
Through the active component that said method is prepared into, in white, tasteless, soluble in water, be dissolved in dimethyl sulfoxide, be slightly soluble in ethanol and methanol, when analyzing with TLC, with chloroform: methanol=1:1 is developing solvent, be on 5cm forward Silica plate at height, its Rf value is about 0.5-0.6, and this sample has significant ultraviolet absorption peak under 210nm wavelength, and the content in magnificent floss bud rattan is 0.05 ‰.
embodiment 6: embodiment 5 magnificent floss bud rattan extract active component activates p53
-/-
gFP fluorescence experiments in-p53-GFPMEF cell, specific experiment operation is with embodiment 2:
experimental result:see Fig. 4,5, under the concentration of 20 μ g/ml, after 72 hours, magnificent floss bud rattan extract active component obviously can activate p53
-/-the expression of GFP fluorescence in-p53-GFPMEF cell; In Fig. 4, A, B are respectively Differential Interference Contrast figure DIC and the GFP fluorogram of negative control 1%DMSO; In Fig. 5, A, B are respectively Differential Interference Contrast figure DIC and the GFP fluorogram of drug treating 20 μ g/ml China floss bud rattan extract active component.
embodiment 7: embodiment 5 magnificent floss bud rattan extract active component has obvious lethal effect to tumor cell, more weak to normal cell (WT-MEF cell) lethal effect
specific experiment operation is with embodiment 3:
Experimental result (table 2): magnificent floss bud rattan extract active component has significant growth inhibitory activity to tumor cell, A549 cell IC
50be 3.67 ± 0.29 μ g/ml, HT29 cell IC
50be 3.68 ± 0.08 μ g/ml, more weak to the suppression of WT-MEF cell, IC
50be 15.47 ± 0.26 μ g/ml, illustrate that this active component all has potential antitumor action to the tumor cell that p53 is wild type and saltant type, more weak to normal cell (WT-MEF cell) lethal effect.
Table 2: the selectivity anti-tumor activity result of magnificent floss bud rattan extract active component
。
embodiment 8: embodiment 5 magnificent floss bud rattan extract active component increases the expression of p53 albumen in A549 cell
specific experiment operation is with embodiment 4:
Experimental result: compared with DMSO matched group, magnificent floss bud rattan extract active component obviously can increase the expression of p53 albumen in A549, and has certain concentration dependent (Fig. 6).
Sequence table
<110> Kunming University of Science and Technology Yunnan Province Agriculture Academy of Science Medicine Plant Institute
The preparation method of <120> mono-kind magnificent floss bud rattan extract and application
<160>2
<170>PatentInversion3.3
<210>1
<211>25
<212>DNA
<213> artificial sequence
<400>1
tggctcgaggtctttacagagagtg25
<210>2
<211>23
<212>DNA
<213> artificial sequence
<400>2
cgagatctcggagaagcgtgaca23
Claims (2)
1. a preparation method for magnificent floss bud rattan extract, is characterized in that carrying out as follows:
(1) pulverizing and lixiviate
After plant China floss bud rattan is pulverized, with pharmaceutical grade methanol room temperature lixiviate 4-5 days, sucking filtration, filtrate concentrates, and obtains methanol extract study, by ethyl acetate and water extract and separate, obtains water layer extractum;
(2) abstraction and purification
By water layer extractum through non-polar macroporous resin pillar layer separation, solvent system is ethanol-water mixture, collects and merges ethanol: water volume ratio is the elution fraction of 8:2, concentrated, obtain magnificent floss bud rattan crude extract, by crude extract water dissolution, methanol extraction, filter, and precipitate 3 times by methanol wash, precipitate through ODS pillar layer separation, solvent system is methanol-water mixture, collect and merge methanol: water volume ratio is the elution fraction of 8:2, concentrated, obtain magnificent floss bud rattan extract.
2. the extract that described in claim 1, the preparation method of magnificent floss bud rattan extract obtains is preparing the application in antitumor personalized treatment medicine.
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