CN103948746B - Application of the Chinese Stellera Root in medicine for anti transfer of tumor is prepared - Google Patents

Abstract

It is an object of the invention to provide application of the Chinese Stellera Root in the medicine for preparing anti-tumor metastasis.Chinese Stellera Root of the present invention, both can be Chinese Stellera Root medicinal material or stellera chamaejasme L extract, or monomer component.

Description

Application of the Chinese Stellera Root in medicine for anti transfer of tumor is prepared

Technical field

The invention belongs to pharmaceutical field, is related to Chinese Stellera Root or its extract in antitumor and anti-tumor metastasis medicine Effect.

Background technology

Malignant tumour is that today's society threatens human health and the major disease disease of survival of patients.For malignant tumour Medicine research and development also turn into the research emphasis and difficult point of current field of medicaments.In recent years, the intervention target spot of anti-tumor medicine Huge progress and perfect is all achieved with drug therapy theory.The particularly research and development of fluorescent dye with tumour-specific targeting medicine, greatly The validity of anticancer is improved, also causes the treatment of patient and outcome to obtain very big improvement.But malignant tumour is invaded It is the maximum killer for threatening tumor patient existence all the time to attack and shift, while it also turns into the maximum bottleneck of oncotherapy.Working as In modern miscellaneous cancer therapy drug market, the chemotherapeutics of a clinical line（Taxol, adriamycin, Doxorubicin etc.）Greatly Multiaction is more rare as the medicine of specific target spot using metastases in fields such as the existence of tumour, propagation, angiogenesis, Simultaneously in medicine is ground, the starting stage theoretical is also in for the mentality of designing of action target spot and just with tumor invasion-shift.Cause This, urgent clinical demand and weak medicine support this contradiction, turn into the biggest obstacle of oncotherapy now, also reflect Direction and trend as future tumors medicament research and development.

Chinese Stellera Root (Stellera chamaejasme Linn.) is that the perennial rhizome type draft of Thymelaeceae stellera is planted Thing, it is distributed widely in the ground such as northeast, North China, northwest.Medicinal material obtains and prepared extremely economical convenient.Meanwhile the root of langdu is used as medicine earliest 《Sheng Nong's herbal classic》In have it is described.Chinese Stellera Root has dissipating bind in the traditional Chinese medical science, relieved oedema or abdominal distension through diuresis or purgation and other effects;Its clinical practice is cured in Gu Book《Sheng Nong's herbal classic》、《Collect efficacious prescriptions》、《The southern regions of the Yunnan Province book on Chinese herbal medicine》Also it is [6] on the books in.Chinese Stellera Root treatment tumor section has Deep experience of tcm inflammation and good theory of traditional Chinese medical science support that first, it kills tumour, is combatting poison with poison based on the traditional Chinese medical science The rules for the treatment of.He also has the function [7] of " eliminating the phlegm of relieving oedema or abdominal distension through diuresis or purgation " effect and " righting " simultaneously, therefore is also based on the allusion quotation of " phlegm syndrome " treatment Model.

Based on this, modern Chinese herbal medicine research provides completely newly for Chinese Stellera Root anticancer as the feasibility of anticancer drug candidate With believable evidence, the theory of traditional Chinese medical science of root of langdu anticancer is further supported and proved.

First, chemical analysis shows, it mainly contains the chemical combination such as Diterpenes, flavonoids, Coumarins and lignanoids Thing, in its root also containing sterol, triterpene, resin and poisonous polymeric organic acid.In above-mentioned many extracts, portion Packet point is proved to have clear and definite active anticancer.

What is more important, pharmaceutical research show that Chinese Stellera Root can be by diversified approach killing tumor cell. On the one hand, he has stronger tumor cytotoxicity（Cytotoxicity effects）Effect, while tumour cell can also be suppressed Cycle and division growth；On the other hand, Chinese Stellera Root also has certain regulatory function to host immune system, so as to from swollen The angle of knurl-host's interaction, evidence is provided for the antitumor action of Chinese Stellera Root.

In all age, though the anticancer function of Chinese Stellera Root has obtained higher attention, its relatively high poison in the application Side effect significantly limit its extensive use, also as the maximum predicament of current root of langdu drug effect and pharmaceutical research.And dropping After low dosage and Drug level, the drug effect of its killing tumor cell substantially reduces again.Therefore, put forth effort to solve in root of langdu application " poison-effect relation ", how ensure drug safety less toxic dosage range in, to greatest extent retain root of langdu extract medicine Effect, the efficient progress for suppressing tumor disease, alleviate the disease burden of drug user, be the unique effective of solution root of langdu application problem Resolution policy, also turn into the wide variety of point of penetration of the root of langdu and break-through point naturally.

Urgent demand of the invention based on clinical cancer therapy, with what is prompted with good theories integration and Primary Study Chinese Stellera Root is research object, and the extraction and identification to its anticancer component have carried out comprehensive and brand-new analysis and research.And low In the dosage range of poison, proved by pharmacodynamics identification and Pharmacological Analysis, stellera chamaejasme L extract and the part wherein included Monomer component（Such as ICJ）There is the drug effect for clearly suppressing tumor motion-transfer in high invasiveness breast cancer, and pass through Proved first in body and isolated experiment, it can be using TGF-beta as internal drug target, by suppressing tumour cell EMT processes, effectively suppress tumour local infiltration and distant metastasis.The present invention is by the binding mode of Chinese Stellera Root from high agent The target spot that amount, high toxicity, tumour existence suppress is transferred to low dosage, hypotoxicity, the target spot of tumor motion regulation.So as to by changing The method for becoming drug target and binding mode effectively reduces Chinese Stellera Root toxic side effect, and is shifted for clinical tumor Intervention this treatment predicament provide effective thinking and direction；And provided for its following medicament research and development and clinical test Believable theories integration and basic research evidence.

The present invention is, as research object, to observe the work of Chinese Stellera Root and monomer to persister with tumor drug resistance cell line With research shows that Chinese Stellera Root can be blocked with inducible resistance strain generating period, and then apoptosis occurs, while stock path AKT lives Change and reduce, there is good lethal effect to drug-resistant cell strain.

The content of the invention

It is an object of the invention to provide application of the Chinese Stellera Root in the medicine for preparing anti-tumor metastasis.

Chinese Stellera Root of the present invention, both can be Chinese Stellera Root medicinal material or stellera chamaejasme L extract, or Monomer component.

Chinese Stellera Root of the present invention or its extract or monomer component have regulation TGF-beta function balance, promote Enter the drug activity of Remolding of Functions of the TGF-beta in tumour cell.

There is up-regulation to suppress oncogene and epithelial molecules core for Chinese Stellera Root of the present invention or its extract or monomer component Heart mark CDH1 expression, suppression interstitial core molecular marker VIM expression, the EMT processes of modulate tumor cell, Suppress the interstitialization transformation of tumour cell, recover the effect of the epithelium attribute of tumour cell from molecular level.

Chinese Stellera Root of the present invention or its extract or monomer component have the tumour cell for blocking TGF-beta inductions The enhancing of locomitivity and invasive ability, maintain tumour cell under TGF-beta inductions migration-invasive ability in reduced levels, So as to the facilitation from functional level antagonism TGF-beta to tumor cell migration-invasive ability.Meanwhile it is horizontal in vivo, The transition intensity of the high transfer strain of breast cancer can substantially be suppressed.

Chinese Stellera Root of the present invention or its extract or monomer component have the tumour for reversing and suppressing TGR-beta inductions Cell EMT processes, the expression for the tumour cell epithelial molecules core mark CDH1 being maintained under TGF-beta inductions, suppression The interstitial core molecular marker VIM of TGF-beta inductions processed up-regulated expression, so as to which antagonism TGF-beta is to tumour cell EMT The induction and influence of process.

Stellera chamaejasme L extract of the present invention both can commercially be bought, and can also be added by conventional technical means Work obtains, including：Crushing, squeezing, calcining, grinding, sieving, diacolation, extraction, water extraction, alcohol extracting, ester carries, ketone carries, the methods of chromatographing Obtain.

Stellera chamaejasme L extract of the present invention, it can be prepared by the following method to obtain：

Using the ethanol extract of Chinese Stellera Root medicinal material 95% as raw material, first step normal phase column chromatography uses 200~300 mesh silica gel, Second step reversed phase column chromatography uses 40~50 μm of ODS, and prepared by the 3rd step high performance liquid chromatography is prepared using 5 μm of C18 Bonded Phases Post, the methanol-water solution of volumetric concentration 75% is used as eluent system, 6 kinds of pure compounds are obtained through the separation of three steps.

It is as follows according to one of embodiment, step of preparation process：

（1）Normal-phase silica gel column chromatography：Take the ethanol of Chinese Stellera Root medicinal material 95% to extract medicinal extract, with one kind in chloroform, methanol or Two kinds of mixed liquor dissolving, in the case where being stirred continuously, 00 mesh column chromatography silica gel of the quality such as addition and medicinal extract, sample is mixed, is filled under normal temperature Divide and dry；Dry method loading afterwards, normal pressure descended the silicagel column of 200~300 mesh 200~3, successively with chloroform, chloroform-methanol 100：1、 Chloroform-methanol 50：1st, chloroform-methanol 20：1 elution, collects chloroform-methanol 20：1 fraction, after negative pressure concentration and recovery solvent, obtain One-level rough segmentation thing.

（2）Anti-phase ODS column chromatographies：After above-mentioned one-level rough segmentation thing is dissolved with methanol, the quality such as addition and one-level rough segmentation thing Particle diameter mix sample for 40~50 μm of ODS column chromatography fillers, fully dried under normal temperature and handle powdering；Dry method loading afterwards, mistake 40~50 μm of ODS chromatographic columns（Column length 40cm, internal diameter 8cm）, medium lift pump pressurization, pressure be 5~10MPa, flow controls 50~ 100ml/min, eluted successively with 15%, 30%, 45%, 60%, 70% methanol-water, collect 70% methanol-water fraction, negative pressure concentrates back After receiving solvent, two level rough segmentation thing is obtained.

The stellera chamaejasme L extract of the present invention both can be one-level rough segmentation thing or two level rough segmentation thing.

Chinese Stellera Root monomer of the present invention, can be following component：

Isodaphnoretin, wild goose skin element D, wild goose skin element C,（+）- the root of langdu peaceful B, the different peaceful B of the root of langdu, the peaceful E of the root of langdu.

It is a further object of the invention to provide contain Chinese Stellera Root or the drug regimen of its extract or monomer component Application of the thing in the medicine for preparing anti-tumor metastasis.

It is still another object of the present invention to provide contain Chinese Stellera Root or the drug regimen of its extract or monomer component Application of the thing in the medicine for preparing anti-anti-drug resistance.

The pharmaceutical composition of the present invention can be any pharmaceutically useful formulation, and these formulations include：Tablet, sugar coated tablet, Film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, mouth containing agent, granule, electuary, ball Agent, powder, paste, sublimed preparation, supensoid agent, pulvis, solution, injection, suppository, ointment, emplastrum, creme, spray, drop Agent, patch.The preparation of the present invention, preferably peroral dosage form, such as：Capsule, tablet, oral liquid, granule, pill, powder, Sublimed preparation, paste etc..Most preferably capsule.

The pharmaceutical composition of the present invention, its preparation being administered orally contain conventional excipient, such as adhesive, filling Agent, diluent, tablet agent, lubricant, disintegrant, colouring agent, flavor enhancement and wetting agent, tablet can be coated if necessary.

Applicable filler includes cellulose, mannitol, lactose and other similar fillers.Suitable disintegrant bag Include starch, polyvinylpyrrolidone and starch derivatives, such as sodium starch glycollate.Suitable lubricant includes, such as firmly Fatty acid magnesium.Suitable pharmaceutically acceptable wetting agent includes lauryl sodium sulfate.

Solid oral composition can be prepared by conventional methods such as mixing, filling, tablettings.Work can be made by carrying out mixing repeatedly Property material be distributed in entirely using a large amount of fillers those compositions in.

The form of oral liquid for example can be water-based or oily suspensions, solution, emulsion, syrup or elixir, Or can be a kind of dry products that water or other suitable carriers can be used to compound before use.This liquid preparation can contain There are conventional additive, such as suspending agent, such as sorbierite, syrup, methylcellulose, gelatin, hydroxyethyl cellulose, carboxymethyl Cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agent, such as lecithin, anhydro sorbitol monooleate or I Primary glue；Non-aqueous carrier（They can include edible oil）, such as the oiliness of the ester of apricot kernel oil, fractionated coconut oil, such as glycerine Ester, propane diols or ethanol；Preservative, such as para hydroxybenzene methyl esters or propylparaben or sorbic acid, and if need Will, contain conventional flavouring agent or colouring agent.

For injection, the fluid unit dosage form of preparation contains the active material and sterile carrier of the present invention.According to carrier And concentration, this compound can be suspended or be dissolved.The preparation of solution is dissolved in a kind of load typically by by active material In body, sterilization is filtered before a kind of suitable bottle or ampoule is loaded into, is then sealed.For example a kind of local anaesthesia of auxiliary material Agent, preservative and buffer can also be dissolved in this carrier., can be after bottle be loaded by this in order to improve its stability Kind composition frost, and under vacuo remove water.

The pharmaceutical composition of the present invention, suitable pharmaceutically acceptable load is optionally added when being prepared into medicament Body, the pharmaceutically acceptable carrier are selected from：Mannitol, sorbierite, sodium pyrosulfite, sodium hydrogensulfite, sodium thiosulfate, salt Sour cysteine, TGA, methionine, injection Vitamin B_6 DTA disodiums, Ethylenediaminetetraacetic Acid Calcium Salt, carbonate, the acetic acid of monovalence alkali metal Salt, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, wheat Bud sugar, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, cellulose and its Derivative, alginates, gelatin, polyvinylpyrrolidone, glycerine, POLYSORBATE 80, agar, calcium carbonate, calcium bicarbonate, surface-active Agent, polyethylene glycol, cyclodextrin, beta-schardinger dextrin, phospholipid material, kaolin, talcum powder, calcium stearate, magnesium stearate etc..

The medicinal material plant distributions of the present invention are extensive, while the mature preparation process of our extracts for being established and monomer is steady It is fixed, it is quality controllable reliable.

Brief description of the drawings

Fig. 1 a：Using the high invasiveness breast cancer MDA-MB-231 of people as cell model, it was demonstrated that stellera chamaejasme L extract is at low dose In the range of amount, there is extremely faint in vitro toxicity, the growth and proliferative effect to cell do not have significant difference.

Fig. 1 b：Using the high invasiveness breast cancer 4T1 of mouse as cell model, it was demonstrated that stellera chamaejasme L extract is in low dosage scope It is interior, there is extremely faint in vitro toxicity, the growth and proliferative effect to cell do not have significant difference.With high invasiveness breast Gland cancer 4T1, MDA-231 is model, using MTT experiment as detection means, the results showed that low dosage stellera chamaejasme L extract and monomer The influence of existence and multiplication capacity of the component to cell is more faint, prompts at the dosage in what safe-dosaging limits.

Fig. 2：It is detection hand with Transwell experiments and Matrigel experiments using high invasiveness breast cancer 4T1 as model Section, the results showed that low dosage stellera chamaejasme L extract and monomer component ICJ can substantially suppress the motion Invasion Potential of tumour cell.

Fig. 3：In vivo studies research shows that low dosage ICJ is more faint on mouse weight influence, no difference of science of statistics, carries Show that it locates that what is nontoxic, less toxic dosage range in vivo, security can ensure.

Fig. 4 a：Using the high invasiveness breast cancer of mouse of luciferase mark as disease model, the mammary gland in situ of mouse is carried out Lotus knurl simultaneously carries out pharmaceutical intervention.In vivo studies research shows that low dosage ICJ can partly suppress the life of mouse breast cancer in situ It is long.Toy images are derived from after mouse administration 25 days in figure.

Fig. 4 b：After tumor-bearing mice in situ tumor resection, the volume statistics of mouse in situ tumor.

Fig. 4 c：After tumor-bearing mice in situ tumor resection, the statistics of mouse in situ tumor knurl weight.

Fig. 4 d：25 days after tumor-bearing mice administration, the total number of photons of mouse in situ tumor is lived together.

Fig. 5 a：In vivo studies research shows that low dosage ICJ can clearly suppress the transfer of breast cancer tumor-bearing mice,

Small animal imaging photo is derived from Post operation 25 days.

Fig. 5 b：Curve map is vertical using the total number of photons of the imaging of mouse different number of days using mouse Post operation number of days as abscissa Coordinate.

Fig. 6：Using the mice serum of above-mentioned zoopery, by ELISA method, detection different pharmaceutical intervention is to mouse Internal TGF-beta expression and the influence of secretion level.As a result show, ICJ being capable of the dose-dependent suppression in the range of low dosage TGF-beta level in Mice Body.

Fig. 7：Western blot test result indicates that, using MDA-231 as cell model, low dosage ICJ processing being capable of agent The up-regulation CDH1 relied on expression is measured, suppresses VIM expression, and then it is disclosed to tumour cell EMT processes from molecular level Effective Regulation effect.

Fig. 8 a：Confocal laser scanning microscope experiment shows, low dosage ICJ processing, can effectively reduce F-actin and exist Intracellular tissue, reduce the polymerization of its fibers form.TGF-beta is reversed to act on F-actin polymerisation induced.

Fig. 8 b：Confocal laser scanning microscope, which is tested, to be shown, low dosage ICJ processing, can effectively induce CDH1 film Positioning, reverse film obscission and intracytoplasmic aggregation caused by TGF-beta.

Fig. 9 a：Transwell test result indicates that, ICJ can from functional level reverse TGF-beta it is thin to MDA-231 The activation of born of the same parents' locomitivity.Clearly suppressing regulation and control of the EMT processes on cell motility influences.

Fig. 9 b：The quantificational expression of Transwell results.

Figure 10：Western Blot test result indicates that, ICJ can from molecular level reverse the non-classical paths of TGF-beta Respectively it is primarily involved in the expression and phosphorylation level of member.Prove that clearly suppression of the ICJ to the non-classical paths of TGF-beta is made With.It is disclosed by suppressing non-classical path, suppresses TGF-beta expression and the activation to EMT processes, further suppression The pharmacological Mechanism of Tumor Cell Migration-invasion and attack-transfer processed and behavior.

Embodiment

By specific examples below, the present invention is further illustrated, but not as limitation of the present invention.

Embodiment 1, the preparation method of stellera chamaejasme L extract

In the present embodiment, using the ethanol extract of Chinese Stellera Root medicinal material 95% as raw material, first step normal phase column chromatography uses

200~300 mesh silica gel, second step reversed phase column chromatography use 40~50 μm of ODS, and the 3rd step high-efficient liquid phase color is composed It is standby to prepare post using 5 μm of C18 Bonded Phases, the methanol-water solution of volumetric concentration 75% is used as eluent system, is through the separation of three steps Obtain 6 kinds of pure compounds.Concrete technology step is as follows：

（1）Normal-phase silica gel column chromatography：Take the ethanol of Chinese Stellera Root medicinal material 95% to extract medicinal extract, with one kind in chloroform, methanol or Two kinds of mixed liquor dissolving, in the case where being stirred continuously, 00 mesh column chromatography silica gel of the quality such as addition and medicinal extract, sample is mixed, is filled under normal temperature Divide and dry；Dry method loading afterwards, normal pressure descended the silicagel column of 200~300 mesh 200~3, successively with chloroform, chloroform-methanol 100：1、 Chloroform-methanol 50：1st, chloroform-methanol 20：1 elution, collects chloroform-methanol 20：1 fraction, after negative pressure concentration and recovery solvent, obtain one Level rough segmentation thing.

（2）Anti-phase ODS column chromatographies：After above-mentioned one-level rough segmentation thing is dissolved with methanol, the quality such as addition and one-level rough segmentation thing Particle diameter mix sample for 40~50 μm of ODS column chromatography fillers, fully dried under normal temperature and handle powdering；

Dry method loading afterwards, cross 40~50 μm of ODS chromatographic columns（Column length 40cm, internal diameter 8cm）, medium lift pump pressurization, pressure is 5~10MPa, flow are controlled in 50~100ml/min, eluted successively with 15%, 30%, 45%, 60%, 70% methanol-water, collect 70% Methanol-water fraction, after negative pressure concentration and recovery solvent, obtain two level rough segmentation thing.

The preparation and purification of embodiment 2, extraction components monomeric compound；

It is prepared by high performance liquid chromatography：Above-mentioned two level rough segmentation thing is dissolved with DMSO, concentration is configured to and supplies examination for 70mg/ml Product solution, through 0.45 μm of filtering with microporous membrane；Chromatographic column is that Chromatorex C18 prepare post, 5 μm of particle diameter, size 500 × 60mm；Sampling volume is 1.4ml, using volumetric concentration be 70% methanol-water solution as eluent system, flow control in 60ml/min, Online ultraviolet detection, Detection wavelength are set as 295nm, collect the fraction containing compound 1,2,3,4,5,6, negative pressure drying respectively 6 kinds of light brown powders are obtained, as purity is more than 98% sterling of compound 1,2,3,4,5,6（Quality about 5.8mg successively, 14.1mg, 5.8mg, 23.5mg, 20.4mg, 25.8mg）.

The chemical identification of extraction components and monomeric compound

The physical parameter and Structural Identification result of above-mentioned 6 kinds of compounds are as follows：

Compound 1, white powder.UV（MeOH,）λmaxnm：205,350.MS（+ESI）m/z：353.068（[M+H]+）. Molecular weight 352, molecular formula C19H12O7.The compound identification is Isodaphnoretin.

Compound 2, light brown powder.UV（MeOH,）λmaxnm：215,295.CD（C=0.1mg/ml, MeOH）Non- appearance. MS（+ESI）m/z：557.148（[M+H]+）.Molecular weight 556, molecular formula C31H24O10.The compound is Novel isomeric, life Entitled wild goose skin element D.

Compound 3, light brown powder.UV（MeOH,）λmaxnm：215,295.CD（C=0.1mg/ml, MeOH）Non- appearance. MS（+ESI）m/z：557.148（[M+H]+）.Molecular weight 556, molecular formula C31H24O10.The compound identification is wild goose skin element C。

Compound 4, light brown powder.UV（MeOH,）λmaxnm：215,295.CD（C=0.1mg/ml, MeOH）-4.2 （346）, 0（325）,+13.8（302）,+1.0（274）,+3.2（253）, 0（240）, -2.7（233）, -0.3（225）, -7.3 （217）.=+121.（C=0.1mg/ml, MeOH）.MS（+ESI）m/z：571.165（[M+H]+）.Molecular weight 570, molecular formula are C32H26O10.The compound identification is（+）The peaceful B of-root of langdu.

Compound 5, light brown powder.UV（MeOH,）λmaxnm：215,295.CD（C=0.2mg/ml, MeOH）Non- appearance. MS（+ESI）m/z：571.165（[M+H]+）.Molecular weight 570, molecular formula C32H26O10.The compound identification is the different root of langdu Peaceful B.

Compound 6, light brown powder.UV（MeOH,）λmaxnm：215,295.CD（C=0.1mg/ml, MeOH）-13.0 (309), 0 (299) ,+19.4 (286), 0 (260), -1.1 (254), 0 (247) ,+1.6 (241), 0 (234), -14.1 (217). MS（+ESI）m/z：571.165（[M+H]+）.Molecular weight 570, molecular formula C32H26O10.The compound is Novel isomeric, life The entitled peaceful E of the root of langdu.

Outer Pharmacodynamics screening and identification inside embodiment 3, Chinese Stellera Root extraction components and monomer component

Stellera chamaejasme L extract described in experiment is two level rough segmentation thing in embodiment 1.

Chinese Stellera Root monomer component described in experiment（ICJ）For the peaceful B of (+)-root of langdu.

1 using high invasiveness breast cancer as disease model, and Chinese Stellera Root is demonstrated to tumour cell survival in vitro by mtt assay Safe dose and toxicity dose section, for low toxicity, safety stellera chamaejasme L extract research evidence is provided.

It is cell membrane to choose the high invasiveness breast cancer MDA-MB-231 cells of people and the high invasiveness breast cancer 4T1 cells of mouse Type.Spread with the density of 3000 cells/wells into 96 orifice plates, and drug-treated was carried out with 24 hours after bed board, use various concentrations Stellera chamaejasme L extract handles tumour cell（ESC1ug/ml and ICJ0.5ug/ml）, and in different time points harvesting（Medicine 24/48/72 hour after thing processing）, the existence of cell is detected by mtt assay, breeds level（With the OD570 values and cell of cell Survival rate meter）.

Experimental study shows, the stellera chamaejasme L extract processing of low dosage, can not substantially reduce drug-treated group tumour The absorption values of cell, tumour cell existence and the influence of multiplication capacity do not have significant difference.Therefore the experiment proves body The stellera chamaejasme L extract of outer low dosage does not have clear and definite drug toxicity, and dosage safety is reliable.

2 using high invasiveness breast cancer as disease model, is tested by Transwell, Matrigel is tested, scratch removal is real Test, it was demonstrated that low dosage stellera chamaejasme L extract can be front lower in the premise for not influenceing cell propagation and existence, effectively suppresses tumour The motion of cell, invasive ability.

In Transwell experiments, the high invasiveness breast cancer MDA-MB-231 cells of people and the high invasiveness breast of mouse are chosen Gland cancer 4T1 cells are cell model.Cell is spread into 12 orifice plates in advance with 1*105 cell number, spread into latter 24 hours in right Drug-treated, system for handling selection serum free medium are carried out in number growth period.Extraction components mixture medicines concentration for the treatment of is set It is set to 1ug/ml, monomeric compound drug-treated concentration is set as 0.5ug/ml.After drug-treated 24 hours, vitellophag, with The density of 50000/ cells/well spreads each group cell in 24 hole transwell cells.Transwell upper stratas cell culture body It for serum free medium, bottom chamber cultivating system is 10% blood serum medium to be.After spreading cell, trained in cell culture incubator Support 18 hours.After culture terminates, harvest, fixed cell, upper strata is softly removed using swab stick and does not wear theca cell, and uses crystal violet Theca cell is worn by dyeing lower floor.After dyeing, five high power field of view are randomly selected, are counted to wearing theca cell number, and to each group Count results are analyzed.

In Matrigel experiments, high invasiveness breast cancer MDA-MB-231 cells and the high invasiveness breast of mouse are equally chosen Gland cancer 4T1 cells are cell model.Progenitor cells processing and drug-treated are the same as Transwell experimental designs.It is small in Transwell In the cell of room upper strata, spread in advance prior to 4 DEG C into Matrigel glue（To specifications, using culture medium 1:1 dilution colloid）, and with 37 DEG C be incubated 6 hours, make gel agglutination, and spread after aggegation into drug-treated each group cell, it is 80000/ hole to spread into density, spread into After continue in cell culture incubator and cultivate 24 hours.Harvest, fixed, colouring method and result acquisition methods are the same as above-mentioned Transwell experimental methods.

Tested by Transwell, Matrigel experiments experiments prove, low dosage stellera chamaejasme L extract and monomer into Divide treatment group, wear theca cell number and significantly reduce, compared with negative control group, there is clear and definite significant difference.Low dosage winter daphne Root of langdu extract and monomer component can significantly inhibit motion and the migration potential of tumour cell.

3 using the high invasiveness breast cancer 4T1 of mouse as elementary cell model, 4T1 of the structure with luciferase assay Cell line.And the cell construction bearing mouse model is utilized, and by the method for small animal imaging, dynamic evaluation low dosage in vivo The influence and its inhibition to metastatic tumour that Chinese Stellera Root monomer component grows to neoplasm in situ cancer.Experiment shows low dosage Chinese Stellera Root monomer component（ICJ）Can be in the less toxic dosage range for not influenceing mouse weight, it is in situ swollen that part suppresses mouse The growth of knurl, while the obvious transfer for suppressing mouse breast cancer, the result support the pharmacodynamics evidence of experiment in vitro, from internal Level provides Research foundation for its antimetastatic activity.

In vivo in pharmacodynamic evaluation, modeling method is be inoculated in BALB/C mice with the density of 10000/mouse the 4 In being padded to breast.Small animal imaging detection is carried out after model construction one week（100ul fluoresceins sylvite/mouse）, pass through total number of photons Number, mouse is grouped at random, ensures that the total number of photons of mouse does not possess significant difference in each experimental group and control group. After packet, by the method for intraperitoneal injection, medicine body is carried out to mouse according to 0ug/kg30ug/kg300ug/kg drug concentration Interior intervention, once, each drug injection system is 100ul/ mouse for daily injection.Pharmaceutical intervention is carried out continuously 36 days, during which every Small animal imaging, dynamic, the growing state of noninvasive monitoring tumor-bearing mice in situ tumor were carried out every three days.36 days after administration, lead to Surgery excision mouse in situ tumor is crossed, calculates the volume and weight of in situ tumor, while observes the transfering state of breast cancer in situ And intensity（In terms of total number of photons）, and continue administration 25 days according to former drug concentration and medication after primary tumor excision, comment Sentence inhibition of the medicine exposure to metastatic tumour.After 25 days, the internal organs such as mouse, harvest mouse lung, liver are put to death, in case follow-up inspection Look into.

Test result indicates that low dosage ICJ is more faint on mouse weight influence, each drug is the same as negative control group mouse Body weight no difference of science of statistics.Prove that toxicity is more faint inside low dosage ICJ.In less toxic dosage range, ICJ can be in body Inside points suppress the volume and weight of in situ tumor, and part suppresses total number of photons of in situ tumor.Meanwhile ICJ can be at low dose The dose-dependent transfer stove number and transition intensity for suppressing metastatic tumor in the range of amount（In terms of total number of photons）.Therefore, it is real in vivo The growth of in situ tumor can partly be suppressed by identifying the bright less toxic ICJ of confirmation, hence it is evident that suppress the transfer process of tumour, with effective Clear and definite anti-tumor metastasis drug effect.The pharmacological mechanism research of embodiment 4, low dosage stellera chamaejasme L extract and monomer component

1, using the animal experimental model in last point, obtains each group experimental animal serum, using ELISA method, detects medicine TGF-beta expression in thing treatment group blood serum sample, and analyze effect tendency of the drug-treated to TGF-beta.Experiment Proof, stellera chamaejasme L extract and monomer component can effectively suppress the expression of TGF-beta in serum in tumor-bearing mice body.

The method for taking blood by plucking eyeball, obtained from the experimental animal of the title of Part II the 3rd each drug-treated group and Negative control group animal blood serum.And moderately dilute each group serum（Dilution ratio is 1:30）, pass through ELISA using the blood serum sample Method is detected.ELISA testing results show that TGF-beta contents are obvious in each low dosage Chinese Stellera Root group processing animal blood serum Reduce, therefore originally it is demonstrated experimentally that low dosage stellera chamaejasme L extract and monomer component being capable of dose-dependent suppression in vivo TGF-beta expression and secretion.

2 using high invasiveness breast cancer MDA-231 and 4LK cell as model, and being detected by WesternBlot proves low dosage Stellera chamaejasme L extract can be obviously promoted tumor suppressor gene and epithelial molecules core mark CDH1 expression, suppress interstitial Core element mark VIM expression, the EMT processes of modulate tumor cell, suppress the interstitialization transformation of tumour cell, from molecule Level recovers the epithelium attribute of tumour cell.

Cell is spread into 6 orifice plates with 2*105 number, and carries out pharmaceutical intervention, drug-treated concentration in spreading 24 hours into after It is set as 0.1/0.5/1ug/ml ICJ, continues culture 24/48 hour, and in each time point harvesting, cracked using RIPA Liquid extracts total cellular protein extract, and utilizes Westernblot technologies, detects the table of GAP-associated protein GAP in each drug-treated group Up to horizontal and phosphorylation level.

3 using breast cancer MDA-231 and ZR75-1 cell as model, by the method for indirect immunofluorescence, detects winter daphne wolf Influences of the malicious monomer component ICJ to CDH1 and F-actin Subcellular Localizations and form in tumour cell.And it is evaluated to TGF- Beta blocking effect.

Using 20ng/mlTGF-beta pre-treatment of tumor cells 24 hours, and with adding low dosage Chinese Stellera Root after processing Monomer component ICJ (1ug/ml), continue medicine and act on 48 hours, in corresponding time point harvesting, pass through indirect immunofluorescence Method detect intracellular CDH1 distribution situation：First antibody selects CDH1 antibody, and antibody dilution ratio is 1:200, second The rabbit secondary antibody of antibody selection FITC marks, dilution ratio 1：200.And DAPI dyeing is carried out after dyeing successfully, dyeing liquor is dense Spend for 100ng/ml.Confocal laser scanning microscope is carried out after dyeing and is taken pictures.

4, using breast cancer MDA-231 cells as model, are tested by Transwell, it was demonstrated that low dosage stellera chamaejasme L extract The enhancing of the Tumor Cell Migration ability of TGF-beta inductions can be substantially blocked, maintains tumour cell under TGF-beta inductions Transfer ability is moved in reduced levels, so that the rush from functional level antagonism TGF-beta to Tumor Cell Migration-transfer ability Enter effect.So as to prove that ICJ drug target molecule is TGF-beta.

Using 20ng/mlTGF-beta pre-treatment of tumor cells 36 hours, and with adding low dosage Chinese Stellera Root after processing Monomer component ICJ (1ug/ml), continue medicine and act on 24/48 hour, in each time point harvesting, carried using RIPA lysates Total cellular protein extract is taken, and utilizes Westernblot technologies, detects the expression water of GAP-associated protein GAP in each drug-treated group Gentle phosphorylation level.

5 using breast cancer MDA-231 cells and ZR75-1 cells as model, and being detected by WesternBlot proves：Low dosage Stellera chamaejasme L extract can substantially reverse the tumour cell EMT processes for suppressing TGR-beta inductions, be maintained at TGF-beta and lure Tumour cell epithelial molecules core mark under leading（CDH1 etc.）Expression, suppress TGF-beta induction interstitial core Molecular marker（VIM etc.）Up-regulated expression.So as to being proved from molecular level, ICJ can antagonism TGF-beta to tumour cell The induction and influence of EMT processes.So as to which further clear and definite ICJ drug target molecule is TGF-beta.

Using 20ng/mlTGF-beta pre-treatment of tumor cells 36 hours, and with adding low dosage Chinese Stellera Root after processing Monomer component ICJ (1ug/ml), continue medicine and act on 24/48 hour, in each time point harvesting, carried using RIPA lysates Total cellular protein extract is taken, and utilizes Westernblot technologies, detects the expression water of GAP-associated protein GAP in each drug-treated group Gentle phosphorylation level.

6 using breast cancer MDA-231 cells as model, cross WesternBlot detections, verify TGF-beta associated signal paths Activation levels.Test result indicates that after ICJ processing tumour cells, each important composition molecule in the non-classical paths of TGF-beta Expression（ITGB3）Phosphorylation level（FAK/p38）Clear and definite holddown is shown, so as to from signal path water Flat, further clear and definite Chinese Stellera Root monomer component ICJ can be using TGF-beta as drug target, by suppressing TGF-beta The activation of non-classical signal path, motion-migration level of tumour cell is further suppress, and then finally, it is suppressed that tumour Invasion and attack and transfer.

Using 20ng/mlTGF-beta pre-treatment of tumor cells 36 hours, and with adding low dosage Chinese Stellera Root after processing Monomer component ICJ (1ug/ml), continue medicine and act on 24/48 hour, in each time point harvesting, carried using RIPA lysates Total cellular protein extract is taken, and utilizes Westernblot technologies, detects the expression water of GAP-associated protein GAP in each drug-treated group Gentle phosphorylation level.

Claims (1)

1. applications of the peaceful B of Chinese Stellera Root monomer component (+)-root of langdu in the medicine for preparing anti-breast cancer transfer.