CN104800294B - The extracting method of anti-tumor Chinese medicine extract and application - Google Patents
The extracting method of anti-tumor Chinese medicine extract and application Download PDFInfo
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Abstract
The invention provides a kind of extracting method of anti-tumor Chinese medicine extract and application, belong to the field of Chinese medicines.The extracting method of the anti-tumor Chinese medicine extract include using Papaveraceae, japanese hylomecon root platymiscium rhizome as raw material, carry out alcohol reflux extraction, and obtained extract solution concentrated, drying, obtain anti-tumor Chinese medicine extract.The anti-tumor Chinese medicine extract can improve the percentage of HT 29 and SW620 cell G1 phase cells, and the ratio of tumour cell S phases and G2 phase cells can be reduced, block with induction HT 29 and SW620 cells generation G1 phases, and suppress the effect of the multiplication capacity of two kinds of tumour cells；In addition, after anti-tumor Chinese medicine extract processing colon cancer cell, can cause caspase 3 and caspase 9 expression quantity of activated form increases, and the expression quantity for promoting apoptosis protein can be improved, reduction suppresses the expression quantity of apoptosis protein.
Description
Technical field
The present invention relates to the field of Chinese medicines, in particular to a kind of anti-tumor Chinese medicine extract and its extracting method and should
With.
Background technology
China is vast in territory, and natural conditions are various, is dispersed with extremely abundant conventional medicament resource, is crude drug in the world
Goods and materials source category at most, cultivation history most long country.And there are many authentic medicinal herbses to have won fame both at home and abroad, and export to more than 100
Countries and regions.
Chinese herbal medicine has materials natural, and effect is steady, and toxic side effect is small to wait the incomparable particular advantages of Western medicine, with society
The progress of meeting, the development of productivity, people increasingly pursue good health and a long life, and modern increasingly turns notice from chemical combination patent medicine
To crude drug, it is desirable to safer effective new drug is developed from crude drug to substitute the former, so as to reduce medicine source disease
Occur.Therefore, development Chinese herbal medicine has not only passed on Chinese nation's elite of 1,100, it may have large market prospect and economy
Benefit.
In addition, Chinese herbal medicine is natural materials, the naturality and bioactivity of various composition are maintained, its composition is easily absorbed
Utilize, it is impossible to absorbed also smoothly to excrete, decomposed in vitro by bacterium etc..Its toxic side effect is small, and animal machine
Body does not develop immunity to drugs；And general chemicals is accumulated in body or extended residual into branch；To animal body have compared with
Big toxic side effect.
Tumour refers to body under the effect of the various tumorigenesis factors, and the neoformation that local organization hyperplasia is formed is mesh
Disease rather refractory in preceding medical domain, and with the increasing of people's operating pressure, the factor such as aggravation of environmental pollution swells
Knurl shows multiple trend.And traditional tumour (malignant tumour) treatment is more using means such as Western medicine, radiotherapy, chemotherapy or operations,
Somewhat expensive, and it is very big to the damage of body；Therefore, therefrom medicinal herbs be starting point there is provided one kind have antineoplastic and its
Preparation method is people's technical problem urgently to be resolved hurrily.
In view of this, it is special to propose the present invention.
The content of the invention
The first object of the present invention is to provide a kind of extracting method of anti-tumor Chinese medicine extract, and this method is with opium poppy
Section, the rhizome of japanese hylomecon root platymiscium are raw material, realize the preparation of anti-tumor Chinese medicine extract.The anti-tumor Chinese medicine extract energy
The percentage of HT-29 and SW620 cell G1 phase cells is enough improved, and tumour cell S phases and G2 phase cells can be reduced
Ratio, blocks, and suppress the effect of the multiplication capacity of two kinds of tumour cells with the induction HT-29 and SW620 cells generation G1 phases
Really.
The second object of the present invention is the purposes for providing the anti-tumor Chinese medicine extract that above-mentioned method is extracted.
The anti-tumor Chinese medicine extract processing colon cancer cell after, can cause activated form caspase-3 and
The increase of caspase-9 expression quantity, and the expression quantity for promoting apoptosis protein can be improved, reduction suppresses apoptosis protein
Expression quantity；Therefore available in the application prepared in antineoplastic.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
A kind of extracting method of anti-tumor Chinese medicine extract, including using Papaveraceae, japanese hylomecon root platymiscium rhizome as raw material,
Alcohol reflux extraction is carried out, and obtained extract solution is concentrated, dried, anti-tumor Chinese medicine extract is obtained.
This extracting method of the present invention, realizes the preparation of anti-tumor Chinese medicine extract, Papaveraceae, japanese hylomecon root platymiscium
Rhizome be rich in substantial amounts of alkaloid, its rhizome have in itself wind-damp dispelling, stop blooding, relieve pain, stimulating the circulation of the blood and cause the muscles and joints to relax, the work(such as eliminating stasis to subdue swelling
Effect.And after refluxing extraction, after concentrating and drying, it is found that its extract has antineoplastic effect, the anti-tumor Chinese medicine
Extract can improve the percentage of HT-29 and SW620 cell G1 phase cells, and can reduce tumour cell S phases and G2
The ratio of phase cell, blocks, and suppress the propagation of two kinds of tumour cells with the induction HT-29 and SW620 cells generation G1 phases
The effect of ability.In addition, the extract also has the expression quantity for improving and promoting apoptosis protein, reduction suppresses Apoptosis egg
The effect of white expression quantity, while can also reduce ERK1/2 signaling pathway proteins activity in colon cancer cell so that colon cancer is thin
The ERK1/2 of phosphorylation and the MEK1/2 of phosphorylation expression quantity just decline in born of the same parents, so as to be led to by suppressing ERK1/2 signal transductions
Road reduces tumor cell proliferation.Moreover, the raw material sources of the extract are extensively, and have no side effect, therefore can be applied to well
In the application and preparation of antineoplastic.
Optionally, the extracting method specifically includes following steps：
1), using Papaveraceae, japanese hylomecon root platymiscium rhizome as raw material, with volumetric concentration be 60-80% and quality is described
The alcohol reflux of 17-21 times of raw material is extracted, and obtains extract solution；
2), the extract solution is concentrated, thick paste is obtained；
3), the thick paste is dried, anti-tumor Chinese medicine extract is obtained.
Using volumetric concentration it is 60-80% and quality is that the ethanol of 17-21 times of the raw material can be as far as possible fully by root
Extracts active ingredients in stem raw material are complete.
Optionally, in step 1) in, the raw material include japanese hylomecon root, split more japanese hylomecon root or sharp one kind split in japanese hylomecon root or
It is a variety of.
The specific plant species of above-mentioned three kinds, the medical value of its rhizome is big, and contained active ingredient is enriched, and therefore, can make
For preferred extraction raw material.
Optionally, in step 1) in, the japanese hylomecon root of weight such as the raw material includes, split japanese hylomecon root more or sharp split japanese hylomecon root.
Three of the above raw material is subjected to refluxing extraction according to identical weight so that the active ingredient of obtained extract
It is abundant comprehensive, with more preferably antitumous effect.
Optionally, in step 3) in, the temperature of the drying is 65-75 DEG C.
Drying operation is carried out in the temperature range, the destruction of active ingredient in concentrate can be reduced, so as to ensure that anti-
The drug effect of tumor extract.
Optionally, in step 3) in, specifically include：After the thick paste is dried, crushed, obtain powdered antitumor
Chinese medical extract.
During application, generally require and anti-tumor Chinese medicine extract is configured to corresponding concentration, therefore, in order to just
In application, crushed after thick paste is dried, to obtain powdered anti-tumor Chinese medicine extract.
Optionally, in step 1) in, specifically include：
A), using Papaveraceae, japanese hylomecon root platymiscium rhizome as raw material, be described by 60-80% and quality of volumetric concentration
The alcohol reflux of 19-21 times of raw material is extracted, and is obtained the first extract solution and first and is extracted the dregs of a decoction；
B), addition volumetric concentration is 60-80% in the described first extraction dregs of a decoction and quality is 17-19 times of the raw material
Ethanol carry out second extraction, obtain the second extract solution and second extract the dregs of a decoction；
C), addition volumetric concentration is 60-80% in the described second extraction dregs of a decoction and quality is 17-19 times of the raw material
Ethanol carry out third time extraction, obtain the 3rd extract solution and the 3rd extract the dregs of a decoction；
D), first extract solution, second extract solution and the 3rd extract solution are merged and obtain extract solution.
Carry out three refluxing extractions to raw material, and after amount of alcohol twice used in refluxing extraction it is less, be so easy to original
Extracts active ingredients in material are complete.
Optionally, also include after step d)：Described 3rd is extracted after the dregs of a decoction are dried and is reclaimed.
After refluxing extraction is finished, due to the wood fibre etc. that can also be remained unchanged in the dregs of a decoction containing a certain amount of effective antitumor composition,
Therefore recycled after the obtained dregs of a decoction are dried, to realize the reasonable utilization of resource, reduce and waste.
Application of the Chinese medical extract that described extracting method is extracted in antineoplastic is prepared.
The Chinese medical extract that described extracting method is extracted is being prepared by raising tumour cell p21 protein expressions, and is pressed down
The activity of Cdk-4/Cdk-6-cyclin D1 compounds processed, makes application of the cell cycle arrest in the antineoplastic of G1 phases.
Compared with prior art, beneficial effects of the present invention are：
(1), the anti-tumor Chinese medicine extract has the percentage for improving HT-29 and SW620 cell G1 phase cells, and energy
The ratio of tumour cell S phases and G2 phase cells is enough reduced, is blocked with the induction HT-29 and SW620 cells generation G1 phases, and press down
Make the effect of the multiplication capacity of two kinds of tumour cells.
(2), the anti-tumor Chinese medicine extract processing colon cancer cell after, can cause activated form caspase-3 and
The increase of caspase-9 expression quantity, and the expression quantity for promoting apoptosis protein can be improved, reduction suppresses apoptosis protein
Expression quantity；Therefore available in the application prepared in antineoplastic.
(3), the anti-tumor Chinese medicine extract can reduce ERK1/2 signaling pathway proteins activity in colon cancer cell so that knot
The ERK1/2 of phosphorylation and the MEK1/2 of phosphorylation expression quantity just decline in colon-cancer cell, so that by suppressing ERK1/2 signals
Signal Transduction Pathways reduction tumour cell increases.
(4), the raw material sources of the anti-tumor Chinese medicine extract extensively, have no side effect, and cost is low, therefore can answer well
For in the application and preparation of antineoplastic.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the accompanying drawing used required in technology description to be briefly described.
The EdU cell proliferation experimental result pictures for the Chinese medical extract that Fig. 1 provides for the present invention；
Fig. 2 is apoptosis rate figure of the Chinese medical extract of the invention provided to HT-29；
Fig. 3 is apoptosis rate figure of the Chinese medical extract of the invention provided to SW-620；
Fig. 4-6 is influence figure of the Chinese medical extract of the invention provided to HT-29 cell cycle；
Fig. 7-9 is influence figure of the Chinese medical extract of the invention provided to SW-620 cell cycle；
After Figure 10 is handled HT-29 and SW-620 for the Chinese medical extract provided with the present invention, apoptosis regulation GAP-associated protein GAP
Expression of results figure；
After Figure 11 is handled HT-29 and SW-620 for the Chinese medical extract provided with the present invention, cell cycle regulating is related
Protein expression result figure；
After Figure 12 is handled HT-29 and SW-620 for the Chinese medical extract provided with the present invention, ERK1/2 signal path eggs
White expression of results figure.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted specific in embodiment
Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, be
The conventional products that can be obtained by commercially available purchase.
The invention provides a kind of extracting method of anti-tumor Chinese medicine extract, it includes planting with Papaveraceae, japanese hylomecon root category
The rhizome of thing is raw material, carries out alcohol reflux extraction, and obtained extract solution is concentrated, dried, and obtains anti-tumor Chinese medicine
Extract.More specifically, it comprises the following steps：
S1：Using Papaveraceae, japanese hylomecon root platymiscium rhizome as raw material, with volumetric concentration be 60-80% and quality is described
The alcohol reflux of 17-21 times of raw material is extracted, and obtains extract solution；
S2：The extract solution is concentrated, thick paste is obtained；
S3：The thick paste is dried, anti-tumor Chinese medicine extract is obtained.
The anti-tumor Chinese medicine extract that this method that the present invention is provided is extracted, it is possible to increase HT-29 and SW620 cells G1
The percentage of phase cell, and the ratio of tumour cell S phases and G2 phase cells can be reduced, with induction HT-29 and SW620
The cell generation G1 phases block, and suppress the effect of the multiplication capacity of two kinds of tumour cells；Therefore it can be very good to be applied to anti-
In the application and preparation of tumour medicine.
Next, with reference to the content of the above, embodiment in detail below is enumerated to extracting method of the present invention.
Embodiment 1
Dry rhizome using japanese hylomecon root Hylomecon japonica (Thunb.) pratl as raw material, using volumetric concentration as
70% and quality for 20 times of the raw material alcohol reflux extract, obtain the first extract solution and first extract the dregs of a decoction；Described
Add that volumetric concentration is 70% and quality is that the ethanol of 18 times of the raw material carries out second extraction in the one extraction dregs of a decoction, obtain the
Two extract solutions and second extract the dregs of a decoction；Addition volumetric concentration is 70% in the described second extraction dregs of a decoction and quality is the original
The ethanol of 18 times of material carries out third time extraction, obtains the 3rd extract solution and the 3rd and extracts the dregs of a decoction；By first extract solution, described
Second extract solution and the 3rd extract solution, which merge, obtains extract solution；The extract solution is concentrated, thick paste is obtained；Will be described thick
Cream is crushed after being dried in 70 DEG C, obtains anti-tumor Chinese medicine extract.
Embodiment 2
To split japanese hylomecon root H.japonica (Thunb.) pratl.Var.dissecta (Franch savat.) Fedde more
Dry rhizome be raw material, using volumetric concentration as 60% and quality be 19 times of the raw material alcohol reflux extract, obtain first
Extract solution and first extracts the dregs of a decoction；Addition volumetric concentration is 60% in the described first extraction dregs of a decoction and quality is the raw material
17 times of ethanol carries out second extraction, obtains the second extract solution and second and extracts the dregs of a decoction；Added in the described second extraction dregs of a decoction
Volumetric concentration is 70% and quality is the ethanol progress third time extraction of 17 times of the raw material, obtains the 3rd extract solution and the 3rd
Extract the dregs of a decoction；First extract solution, second extract solution and the 3rd extract solution are merged and obtain extract solution；By institute
Extract solution concentration is stated, thick paste is obtained；Crushed after the thick paste is dried in 65 DEG C, obtain anti-tumor Chinese medicine extract.
Embodiment 3
Using the sharp dry rhizome for splitting japanese hylomecon root H.japonica (Thunb.) pratl.Var.Subincia Fedde as original
Material, using volumetric concentration as 80% and quality be 21 times of the raw material alcohol reflux extract, obtain the first extract solution and first and carry
Get it filled slag；Addition volumetric concentration is 80% in the described first extraction dregs of a decoction and quality is the ethanol progress of 19 times of the raw material
Second extraction, obtains the second extract solution and second and extracts the dregs of a decoction；Described second extract the dregs of a decoction in add volumetric concentration be 80%,
And quality carries out third time extraction for the ethanol of 18 times of the raw material, obtain the 3rd extract solution and the 3rd and extract the dregs of a decoction；Will be described
First extract solution, second extract solution and the 3rd extract solution, which merge, obtains extract solution；The extract solution is concentrated, obtained
To thick paste；Crushed after the thick paste is dried in 70 DEG C, obtain anti-tumor Chinese medicine extract.
Embodiment 4
With etc. quality japanese hylomecon root Hylomecon japonica (Thunb.) pratl, split japanese hylomecon root H.japonica more
(Thunb.) pratl.Var.dissecta (Franch savat.) Fedde, sharp split japanese hylomecon root H.japonica (Thunb.)
Pratl.Var.Subincia Fedde be raw material, using volumetric concentration as 70% and quality be 20 times of the raw material ethanol return
Stream is extracted, and is obtained the first extract solution and first and is extracted the dregs of a decoction；Described first extract the dregs of a decoction in add volumetric concentration be 70% and
Quality carries out second extraction for the ethanol of 18 times of the raw material, obtains the second extract solution and second and extracts the dregs of a decoction；Described second
Extract and add that volumetric concentration is 70% and quality is that the ethanol of 18 times of the raw material carries out third time extraction in the dregs of a decoction, obtain the
Three extract solutions and the 3rd extract the dregs of a decoction；First extract solution, second extract solution and the 3rd extract solution are merged
Obtain extract solution；The extract solution is concentrated, thick paste is obtained；Crushed after the thick paste is dried in 70 DEG C, obtain anti-swollen
Knurl Chinese medical extract.
Embodiment 5
Dry rhizome using japanese hylomecon root Hylomecon japonica (Thunb.) pratl as raw material, using volumetric concentration as
70% and quality for 20 times of the raw material alcohol reflux extract, obtain the first extract solution and first extract the dregs of a decoction；Described
Add that volumetric concentration is 70% and quality is that the ethanol of 18 times of the raw material carries out second extraction in the one extraction dregs of a decoction, obtain the
Two extract solutions and second extract the dregs of a decoction；Addition volumetric concentration is 70% in the described second extraction dregs of a decoction and quality is the original
The ethanol of 18 times of material carries out third time extraction, obtains the 3rd extract solution and the 3rd and extracts the dregs of a decoction；By first extract solution, described
Second extract solution and the 3rd extract solution, which merge, obtains extract solution；The extract solution is concentrated, thick paste is obtained；Will be described thick
Cream is crushed after being dried in 70 DEG C, obtains anti-tumor Chinese medicine extract.
Application examples 1
Anti-tumor Chinese medicine extract (LAOSHUQI group) that the embodiment of the present invention 5 is provided and with other raw material sources (clasps
And QINGWAQI) extract medicine by HT-29, SW620 cell experiment, Primary Study has been carried out to the IC50 of tumour cell, tie
Fruit shows that LAOSHUQI group extract has good antitumor action, and concrete outcome is referring to table 1.
It is pointed out that in the present invention, shown LAOSHUQI (also known as honey raisin tree seven), it comes as a kind of Chinese medicine
Source, includes the plant of Papaveraceae, japanese hylomecon root category (japanese hylomecon root, split japanese hylomecon root more or sharp split japanese hylomecon root).
The IC50 testing results of the anti-tumor Chinese medicine extract in the different material of table 1 source
Application examples 2
EdU cell proliferation experiments：Carried out with conventional EdU kits, shown in result figure 1, as a result find the traditional Chinese medicine extraction
Thing (LAOSHUQI) has the effect of significantly reduction tumour cell HT-29 propagation.
In Fig. 1, A：Con is compareed；B：5-Fu compares (15umol)；C：LAOSHUQI extract (0.01mg/ml)；D：Mouse
Seven extracts (0.05mg/ml)；Wherein, LAOSHUQI extract is that embodiment 5 is prepared.
Application examples 3
Streaming-Apoptosis：By colon cancer cell (HT-29 and SW-620) respectively with 5 × 105The density inoculation in individual/hole
In 6 orifice plates, 0.01,0.05mg/ml concentration LAOSHUQIs extract (embodiment 5 is prepared) is given after 24h is adherent, is continued
Culture is respectively in two time point collecting cells of 24h and 48h, including cell that is all adherent and suspending.
Cell, 195 μ L of each sample Annexin-FITC combination buffers are washed twice with the PBS solution of precooling
(10mM Hepes/NaOH,140mM NaCl,2.5mM CaCl2) cell is resuspended.
The Annexin-FITC solution (25 μ g/mL) for adding 5 μ L is gently mixed, and room temperature lucifuge is incubated 10min.Centrifugation
5min, abandons supernatant, adds 190 μ L AnnexinV-FITC combinations liquid and cell is resuspended.
The PI solution (250 μ g/mL) for adding 10 μ L is gently mixed, immediately with flow cytomery, wavelength 488nm, work(
Rate 15mW, is handled with instrument software, is drawn shown in apoptosis rate Fig. 2-Fig. 3 of cell.
Wherein, in fig. 2, it is HT-29 for examination cell, and is A：Con is compareed；B：5-Fu compares (15umol)；C：Mouse
Seven extracts (0.01mg/ml)；D：LAOSHUQI extract (0.05mg/ml).In figure 3, it is SW-620, A for examination cell：con
Control；B：5-Fu compares (15umol)；C：LAOSHUQI extract (0.01mg/ml)；D：LAOSHUQI extract (0.05mg/ml).
It was found from Fig. 2 and Fig. 3, this anti-tumor Chinese medicine extract that provides of the present invention its there is the effect for significantly causing Apoptosis
Really.
Application examples 4
To the HT-29 after the processing of the LAOSHUQI extract (embodiment 5 is prepared) of 0.01,0.05mg/ml concentration and
SW620 cells carry out PI dyeing, pass through the influence of Drug by Flow Cytometry cell cycle.
(region shown in the black in abscissa leading portion is G1 phase cells, region shown in back segment black shown in result figure 4-9
For G1 phases cell), wherein, Fig. 4-Fig. 6 is HT-29 for examination cell；A：Con is compareed；B：LAOSHUQI extract (0.01mg/
ml)；C：LAOSHUQI extract (0.05mg/ml)；Fig. 7-9 is SW620 cells for examination cell；A：Con is compareed；B：LAOSHUQI is carried
Take thing (0.01mg/ml)；C：LAOSHUQI extract (0.05mg/ml)；From Fig. 4-9, compared with Normal group, the G1 phases are thin
The percentage of born of the same parents is significantly raised after LAOSHUQI extract-treated, point out the concentration Chinese medical extract can induce HT-29 and
The SW620 cells generation G1 phases block.In addition, the multiplication capacity of the ratio reduction prompting cell of S phases and G2 phase cells is received always
The suppression of the extract of mouse seven.
Application examples 5
1) it is administered：HT29 is inoculated with respectively, and SW620 cells are in 6 orifice plates, per hole 2 × 105Given after individual cell, culture 24h
LAOSHUQI extract (embodiment 5 is provided), continues to be incubated 24/48h.
2) cell/tissue total protein is extracted：After cell administration 24/48h, nutrient solution is exhausted, is rinsed 3 times with the PBS of precooling,
Scrape immigration 1.5mL centrifuge tubes after cell.4 DEG C, 3000rpm centrifugation 5min abandon supernatant.
60 μ L RIPA protein lysates (PMSF containing 10mg/mL, instant to match somebody with somebody) are added into centrifuge tube, it is quiet in ice bath
Cracking 30min is put, after cell is fully cracked, 4 DEG C, 12000rpm centrifugation 20min take supernatant stand-by.
3) protein quantification：Protein concentration in cell pyrolysis liquid is determined using BCA methods.The BCA of gradient concentration is prepared first
Standard liquid：1、0.5、0.25、0.125、0.025、0mg/mL.The μ L of standard liquid 10 or the μ L of sample 1 of each concentration are drawn respectively
(adding 9 μ L PBS) then to 200 μ L working solutions are added in hole, fully mixes, after 37 DEG C are incubated 30min into 96 orifice plates
OD values are measured under 570nm wavelength with ELIASA.Standard curve is made to OD values with normal concentration, normal equation is fitted to obtain, according to
Sample OD values calculate its protein concentration.
4) albumen sample preparation：5 × albumen sample-loading buffer of proper volume is added into cell pyrolysis liquid, boiling is placed in after mixing
5min in water.According to protein concentration, electrophoresis applied sample amount is calculated according to the total protein concentration of 20 μ g/ samples.
5) electrophoresis：From 6% concentration glue and 12% or 10% separation gel and carry out protein electrophoresis, by protein sample and
Molecular weight marker respectively after loading with 100V starting voltage by sample concentration to separation gel interface when voltage is adjusted to
120V, untill when bromjophenol blue overflows separation gel.
6) transferring film：NC films are infiltrated in the transfering buffering liquid of precooling.The NC films of moistening are placed in gel surface, both sides are put
The filter paper of wetting is put, bubble is excluded, is placed in after clipping in transferring film groove, the transfering buffering liquid of precooling is added, transfer groove is placed in ice
In bath 70min is transferred under 100V voltages.
7) film is cut：With distilled water by NC film rinsed cleans, the albumen that develops the color is placed in Ponceau S, according to molecular weight of albumen
Marker and required molecular weight of albumen cut out NC films, and film decolouring is washed with PBST.
8) close：NC films are put into the 5% skimmed milk power solution prepared with PBST, film is washed with PBST after room temperature closing 2h
3 times, each 5min.
9) primary antibody is incubated：NC films after cutting out are put into corresponding antibody diluent, 4 DEG C of overnight incubations.
10) secondary antibody is incubated：NC films are washed with PBST 3 times, it is each 5min/ times.1 is pressed with PBST buffer solutions:10000 ratio
The goat antirabbit of horseradish peroxidase-labeled or the secondary antibody of anti-mouse are diluted, 1h is incubated at room temperature.
11) lighted using chemical method, luminous result is taken pictures, statistical analysis is done in OD value scanning.
The experimental result of above-mentioned application examples 5 is mainly reflected in the following aspects：
1st, medicine cell cycle regulates and controls the influence of correlative protein expression
Cysteine proteinase (Caspases) accounts for core status, wherein caspase-8 in apoptosis regulation,
Caspase-9 is the startup enzyme of Apoptosis, and caspases-3 is the main execution enzyme of Apoptosis.
It was found that being handled with Chinese medical extract after colon cancer cell 24h, caspase-3 the and caspase-9 tables of activated form
Start increase up to amount, but the expression quantity of caspase-8 activated forms does not change.
Bcl-2 protein families in apoptosis process there is important regulating and controlling to act on, and Bcl-2 family proteins are divided into two classes：
Promote the albumen of Apoptosis, including Bid, Bax, Bak and Bad etc.；Suppress the albumen of Apoptosis, including Bcl-xl and Bcl-
2。
After LAOSHUQI extract (Chinese medical extract) the processing HT29 and SW620 cells 24/48h of respective concentration, Bax is detected
With the situation of change of Bcl-2 protein expressions, as a result as shown in Figure 10.Western blot testing results show treated with medicaments
Afterwards, HT29 and SW620 cells Bax expressing quantities are significantly raised, while anti-apoptotic proteins Bcl-2 expression substantially drops
It is low, and in concentration and time dependence.
2nd, medicine cell cycle regulates and controls the influence of correlative protein expression
In the regulation and control of cell cycle, cyclin (Cyclin) family and cyclin-dependent kinase (Cdk) family
Race plays an important roll.Cyclin and Cdk combine to form the lasting progress of the kinase complex cell cycle regulation of activation.
Find that the Chinese medical extract inducible colon cancer cell generation G1 phases block by Flow cytometry, the cell cycle
PROTEIN C yclin D1 are combined rear regulating cell with cyclin-dependent kinase Cdk4 and Cdk6 and converted from the G1 phases to the S phases, therefore this
Experiment has looked first at influence of the LAOSHUQI extract to these three protein expressions, as a result finds LAOSHUQI extract-treated cell
After 24/48h, CyclinD1, Cdk4 and Cdk6 expressing quantity is decreased obviously.
P21 is Cdk suppression albumen, and p21 can prevent damage dna from passing through as cell cycle G1 inspection post, suppress by
DNA duplication and accumulation is damaged, so as to play cancer suppressing action.
As shown in figure 11, experiment finds LAOSHUQI of the expression quantity in respective concentration of HT-29 and SW620 cell p21 albumen
Raised after extract-treated.Result above prompting LAOSHUQI extract can suppress Cdk-4/ by raising p21 protein expressions
The activity of Cdk-6-cyclin D1 compounds, makes cell cycle arrest in the G1 phases.
3rd, influence of the medicine to cell MAPK signal paths
The former activated protein kinases of three groups of main mitogens (mitogen-activated proteinkinases,
MAPK) in signaling pathways, ERK1/2 signal transduction pathways have important facilitation to tumor cell proliferation.Therefore, examine
Influence of the Chinese medical extract to ERK1/2 signaling pathway proteins activity in colon cancer cell is surveyed.As a result as shown in figure 12, to
Give after respective concentration drug-treated 30min, the ERK1/2 of the phosphorylation and MEK1/2 of phosphorylation in HT-29 and SW620 cells
Expression quantity begins to decline, and declines level in time dependence, shows that the Chinese medical extract can suppress ERK1/2 signals and turn
The abnormal activation of guiding path, so that with the effect for suppressing tumor cell proliferation.
To sum up, the anti-tumor Chinese medicine extract that the present invention is provided, it is by controlling apoptosis regulation correlative protein expression, regulation and control
The effect for suppressing tumor cell proliferation is realized in terms of cell cycle regulatory protein expression and cell MAPK signal paths,
So as to embody the obvious effect for suppressing tumour, and its raw material sources is extensively, thing side effect, therefore can be very good to be applied to
In antitumor and antineoplastic preparation, with high potential using value.
Although illustrate and describing the present invention with specific embodiment, but it will be appreciated that without departing substantially from the present invention's
Many other changes and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (7)
1. a kind of application of inhibitor against colon carcinoma cells Chinese medical extract in drugs against colon cancer is prepared, it is characterised in that the resistive connection intestines
The extracting method of cancer Chinese medical extract for japanese hylomecon root, split the root of japanese hylomecon root or the sharp one or more plants split in japanese hylomecon root more
Stem is raw material, carries out alcohol reflux extraction, and obtained extract solution is concentrated, dried, and obtains inhibitor against colon carcinoma cells traditional Chinese medicine extraction
Thing.
2. application according to claim 1, it is characterised in that the mechanism of action of the drugs against colon cancer is by up-regulation
Tumour cell p21 protein expressions, and suppress the activity of Cdk-4/Cdk-6-cyclin D1 compounds, make cell cycle arrest in
The G1 phases.
3. application according to claim 1, it is characterised in that the extracting method is specially following steps：
1) japanese hylomecon root or the sharp rhizome for splitting japanese hylomecon root, are split using japanese hylomecon root, as raw material more, is 60-80% and quality with volumetric concentration
Extracted for the alcohol reflux of 17-21 times of the raw material, obtain extract solution；
2), the extract solution is concentrated, thick paste is obtained；
3), the thick paste is dried, inhibitor against colon carcinoma cells Chinese medical extract is obtained.
4. application according to claim 3, it is characterised in that in step 3) in, the temperature of the drying is 65-75 DEG C.
5. application according to claim 3, it is characterised in that in step 3) in, it is specially：
After the thick paste is dried, crushed, obtain powdered inhibitor against colon carcinoma cells Chinese medical extract.
6. the application according to claim any one of 3-5, it is characterised in that in step 1) in, it is specially：
A) japanese hylomecon root or the sharp rhizome for splitting japanese hylomecon root, are split using japanese hylomecon root, as raw material, using volumetric concentration as 60-80% and quality more
Extracted for the alcohol reflux of 19-21 times of the raw material, obtain the first extract solution and first and extract the dregs of a decoction；
B), addition volumetric concentration is 60-80% in the described first extraction dregs of a decoction and quality is the second of 17-19 times of the raw material
Alcohol carries out second extraction, obtains the second extract solution and second and extracts the dregs of a decoction；
C), addition volumetric concentration is 60-80% in the described second extraction dregs of a decoction and quality is the second of 17-19 times of the raw material
Alcohol carries out third time extraction, obtains the 3rd extract solution and the 3rd and extracts the dregs of a decoction；
D), first extract solution, second extract solution and the 3rd extract solution are merged and obtain extract solution.
7. application according to claim 6, it is characterised in that also include after step d)：
Described 3rd is extracted after the dregs of a decoction are dried and is reclaimed.
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|---|---|---|---|---|
| CN1429611A (en) * | 2002-09-13 | 2003-07-16 | 张平 | Greater celandine extract and its preparation method and application |
| CN1579387A (en) * | 2004-05-20 | 2005-02-16 | 北京神农坛医药科技有限公司 | Extraction of protopine from plant, and its manufacture of medicinal preparation and use |
| CN104248635A (en) * | 2013-06-27 | 2014-12-31 | 天津中医药大学 | Application of macleyine alkaloid in preparation of medicine for inhibiting P-gp |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1429611A (en) * | 2002-09-13 | 2003-07-16 | 张平 | Greater celandine extract and its preparation method and application |
| CN1579387A (en) * | 2004-05-20 | 2005-02-16 | 北京神农坛医药科技有限公司 | Extraction of protopine from plant, and its manufacture of medicinal preparation and use |
| CN104248635A (en) * | 2013-06-27 | 2014-12-31 | 天津中医药大学 | Application of macleyine alkaloid in preparation of medicine for inhibiting P-gp |
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| 白屈菜提取工艺的初步研究;刘拉;《广东微量元素科学》;20011231;第8卷(第7期);第57页最后1段 * |
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