CN103948746A - Application of stellera chamaejasme in preparation of anti-tumor metastasis medicaments - Google Patents
Application of stellera chamaejasme in preparation of anti-tumor metastasis medicaments Download PDFInfo
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Abstract
The purpose of the present invention is to provide an application of stellera chamaejasme in preparation of anti-tumor metastasis medicaments. The stellera chamaejasme of the invention can be either stellera chamaejasme medicinal material or stellera chamaejasme extract, or can be a monomer component.
Description
Technical field
The invention belongs to pharmaceutical field, relate to Stellera chamaejasme L. or the effect of its extract in the medicine of antitumor and anti metastasis.
Background technology
Malignant tumor is sick kind of major disease that society threatens human health and survival of patients.For the medicine research and development of malignant tumor, also become research emphasis and the difficult point of current field of medicaments.In recent years, the intervention target spot of anti-tumor medicine and Drug therapy theory have all obtained huge progress and perfect.The particularly research and development of tumour-specific targeted drug, have improved anticancer effectiveness greatly, also make patient's treatment and prognosis effect obtain very big improvement.But the invasion and attack of malignant tumor and transfer are the maximum killer who threatens tumor patient existence all the time, it also becomes the maximum bottleneck of oncotherapy simultaneously.In current miscellaneous cancer therapy drug market, the chemotherapeutics of a clinical line (such as paclitaxel, amycin, doxorubicin etc.) acts on the fields such as the existence, propagation, angiogenesis of tumor mostly, the medicine that the neoplasm metastasis of take is specificity target spot is comparatively rare, simultaneously in grinding medicine, the mentality of designing that the tumor invasion-shift of take is action target spot and theory are also just in the starting stage.Therefore, urgent clinical demand and weak medicine are supported this contradiction, become the biggest obstacle of oncotherapy now, also reflect the direction and the trend that become following tumour medicine research and development.
Stellera chamaejasme L. (Stellera chamaejasme Linn.) is the perennial rhizome type of thymelaeceae stellera herbaceous plant, is distributed widely in the ground such as northeast, North China, northwest.Medical material obtain and prepare very economical convenient.Meanwhile, the Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) is used as medicine and in the > > of < < Sheng Nong's herbal classic, is recorded to some extent.The effect such as Stellera chamaejasme L. has eliminating stagnation in the traditional Chinese medical science, relieve oedema or abdominal distension through diuresis or purgation; Its clinical practice also all [6] on the books in ancient medical book < < Sheng Nong's herbal classic > >, < < collection efficacious prescriptions > >, < < the southern regions of the Yunnan Province book on Chinese herbal medicine > > etc.Stellera chamaejasme L. treatment tumor section has deep experience of tcm inflammation and good theory of Chinese medical science support, and first, its killing tumor cells, is the rule for the treatment of of the treating the poisonous disease with poisonous drugs based on the traditional Chinese medical science.He also has the function [7] of " relieve oedema or abdominal distension through diuresis or purgation and eliminate the phlegm " effect and " righting " simultaneously, is also therefore the model based on " phlegm syndrome " treatment.
Based on this, modern Chinese medicine is learned and is studied as the anticancer feasibility as anticancer drug candidate of Stellera chamaejasme L. provides brand-new and believable evidence, has further supported and proved the anticancer theory of Chinese medical science of the Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae).
First, chemical analysis shows, it mainly contains the compounds such as Diterpenes, flavonoid, Coumarins and lignanoids, at its root, also contains sterol, triterpene, resin and poisonous polymeric organic acid.In above-mentioned many extracts, part component is proved to be has clear and definite active anticancer.
What is more important, pharmaceutical research shows that Stellera chamaejasme L. can be by diversified approach killing tumor cell.On the one hand, he has stronger tumor cytotoxicity (Cytotoxicity effects) effect, cycle and division growth that simultaneously can also inhibition tumor cell; On the other hand, Stellera chamaejasme L. also has certain regulatory function to host immune system, thereby from the interactional angle of tumor-host, for the antitumor action of Stellera chamaejasme L. provides evidence.
In all age, though the anticancer function of Stellera chamaejasme L. has obtained higher attention, but its higher toxic and side effects in application has greatly limited its extensive use, also becomes the maximum predicament of current Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) drug effect and pharmaceutical research.And after reducing using dosage and Drug level, the drug effect of its killing tumor cell reduces again greatly.Therefore, put forth effort to solve " poison-effect relationship " in Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) application, how in guaranteeing the low toxicity dosage range of drug safety, the drug effect that retains to greatest extent Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) extract, suppress efficiently the progress of tumor disease, alleviating medication person's disease burden, is the unique effective resolution policy that solves a Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) application difficult problem, also naturally becomes point of penetration and the break-through point of Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae) extensive use.
The present invention is based on the urgent demand of clinical cancer treatment, take and has good theoretical support and Stellera chamaejasme L. that preliminary study is pointed out is object of study, and the extraction of its anticancer component and evaluation have been carried out to comprehensive and brand-new analysis and research.And in the dosage range of low toxicity, by pharmacodynamics, identify with Pharmacological Analysis and prove, stellera chamaejasme L extract and the partial monosomy component (such as ICJ) wherein comprising have the drug effect of clear and definite inhibition tumor motion-transfer in high aggressivity breast carcinoma, and by proving first at body and isolated experiment, it can take the drug target of TGF-beta in body, by the EMT process of inhibition tumor cell, effectively suppress local infiltration and the distant metastasis of tumor.The present invention suppresses the binding mode of Stellera chamaejasme L. target spot from high dose, high toxicity, tumor existence is transferred to the target spot of low dosage, hypotoxicity, tumor motion adjusting.Thereby by changing the method for drug target and binding mode, effectively reduce Stellera chamaejasme L. toxic and side effects, and this treatment predicament of intervention of shifting for clinical tumor effective thinking and direction are provided; And provide believable theory support and basic research evidence for its following medicament research and development and clinical trial.
The present invention be take and had tumor drug resistance cell strain as object of study, observe Stellera chamaejasme L. and the effect of monomer to persister, studies show that Stellera chamaejasme L. can block by inducible resistance strain generating period, and then generation apoptosis, stock path AKT activation simultaneously reduces, and drug-resistant cell strain is had to good lethal effect.
Summary of the invention
The object of the present invention is to provide the application of Stellera chamaejasme L. in the medicine of preparation anti metastasis.
Stellera chamaejasme L. of the present invention, can be both Stellera chamaejasme L. medical material, can be also stellera chamaejasme L extract, or monomer component.
Stellera chamaejasme L. of the present invention or its extract or monomer component have the function balance that regulates TGF-beta, promote the drug activity of the Remolding of Functions of TGF-beta in tumor cell.
Stellera chamaejasme L. of the present invention or its extract or monomer component have the expression that raises inhibition oncogene and epithelium molecule core mark CDH1, suppress the expression of interstitial core molecular marker VIM, the EMT process of modulate tumor cell, between inhibition tumor cell, materialization changes, and recovers the effect of the epithelium attribute of tumor cell from molecular level.
Stellera chamaejasme L. of the present invention or its extract or monomer component have the Tumor Cell Migration ability of blocking-up TGF-beta induction and the enhancing of invasive ability, maintain tumor cell under TGF-beta induction migration-invasive ability at reduced levels, thereby from functional level antagonism TGF-beta the facilitation to tumor cell migration-invasive ability.Meanwhile, level, can obviously suppress the high transition intensity that shifts strain of breast carcinoma in vivo.
Stellera chamaejasme L. of the present invention or its extract or monomer component have the tumor cell EMT process that suppresses TGR-beta induction that reverses, remain on the expression of the tumor cell epithelium molecule core mark CDH1 under TGF-beta induction, the up-regulated that suppresses the interstitial core molecular marker VIM of TGF-beta induction, thereby induction and the impact of antagonism TGF-beta on tumor cell EMT process.
Stellera chamaejasme L extract of the present invention both can buy on market, also can obtain by routine techniques means processing, comprise: pulverize, squeeze, calcine, grind, sieve, the method such as percolation, extraction, water extraction, alcohol extraction, ester are carried, ketone is carried, chromatography obtains.
Stellera chamaejasme L extract of the present invention, can prepare by the following method:
Stellera chamaejasme L. medical material 95% ethanol extraction of take is raw material, first step normal phase column chromatography adopts 200～300 order silica gel, second step reversed phase column chromatography adopts 40～50 μ m ODS, the 3rd step high performance liquid chromatography preparation adopts 5 μ mC18 Bonded Phase preparative columns, adopting the methanol-water solution of volumetric concentration 75% is eluent system, through three step separation, obtains 6 kinds of pure compounds.
According to one of embodiment, step of preparation process is as follows:
(1) purification on normal-phase silica gel column chromatography: get Stellera chamaejasme L. medical material 95% ethanol extraction extractum, dissolve with one or both the mixed liquor in chloroform, methanol, under constantly stirring, add the 00 order column chromatography silica gel with the quality such as extractum, mix sample, fully dry under room temperature; Dry method loading afterwards, normal pressure descended 200～300 order 200～3 silicagel columns, successively with chloroform, chloroform-methanol 100:1, chloroform-methanol 50:1, chloroform-methanol 20:1 eluting, collected chloroform-methanol 20:1 stream part, after negative pressure concentration and recovery solvent, obtain one-level rough segmentation thing.
(2) anti-phase ODS column chromatography: by above-mentioned one-level rough segmentation thing with dissolve with methanol after, adding with the particle diameter of the quality such as one-level rough segmentation thing is that 40～50 μ m ODS column chromatography fillers are mixed sample, fully dries and be processed into powdery under room temperature; Dry method loading afterwards, cross 40～50 μ m ODS chromatographic column (column length 40cm, internal diameter 8cm), medium lift pump pressurization, pressure is 5～10MPa, flow-control is at 50～100ml/min, successively with 15%, 30%, 45%, 60%, 70% methanol-water eluting, collect 70% methanol-water stream part, after negative pressure concentration and recovery solvent, obtain secondary rough segmentation thing.
Stellera chamaejasme L extract of the present invention can be both one-level rough segmentation thing, can be also secondary rough segmentation thing.
Stellera chamaejasme L. monomer of the present invention can be following composition:
Isodaphnoretin, sikokianin D, sikokianin C, the peaceful B of (+)-Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae), the peaceful B of the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae), the peaceful E of the Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae).
Another object of the present invention is, the application of the pharmaceutical composition that contains Stellera chamaejasme L. or its extract or monomer component in the medicine of preparation anti metastasis is provided.
A further object of the present invention is, the application of the pharmaceutical composition that contains Stellera chamaejasme L. or its extract or monomer component in the medicine of the anti-anti-drug resistance of preparation is provided.
Pharmaceutical composition of the present invention can be any pharmaceutically useful dosage form, and these dosage forms comprise: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, suck agent, granule, electuary, pill, powder, unguentum, sublimed preparation, suspensoid, powder, solution, injection, suppository, ointment, plaster, cream, spray, drop, patch.Preparation of the present invention, peroral dosage form preferably, as: capsule, tablet, oral liquid, granule, pill, powder, sublimed preparation, unguentum etc.Capsule most preferably.
Pharmaceutical composition of the present invention, the preparation of its oral administration can contain conventional excipient, such as binding agent, filler, diluent, tablet agent, lubricant, disintegrating agent, coloring agent, flavoring agent and wetting agent, can carry out coating to tablet if desired.
Applicable filler comprises cellulose, mannitol, lactose and other similar filler.Suitable disintegrating agent comprises starch, polyvinylpyrrolidone and starch derivatives, for example sodium starch glycollate.Suitable lubricant comprises, for example magnesium stearate.The acceptable wetting agent of suitable medicine comprises sodium lauryl sulphate.
Can fill by mixing, the method that tabletting etc. are conventional is prepared solid oral composition.Repeatedly mix and can make active substance be distributed in those compositionss of a large amount of filleies of whole use.
The form of oral liquid can be for example aqueous or oily suspensions, solution, Emulsion, syrup or elixir, or can be a kind of available water before use or the composite dry products of other suitable carrier.This liquid preparation can contain conventional additive, such as suspending agent, for example sorbitol, syrup, methylcellulose, gelatin, hydroxyethyl-cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenation edible fat, emulsifying agent, for example lecithin, anhydro sorbitol monooleate or arabic gum; Non-aqueous carrier (they can comprise edible oil), for example almond oil, fractionated coconut oil, such as oily ester, propylene glycol or the ethanol of the ester of glycerol; Antiseptic, for example para hydroxybenzene methyl ester or propyl p-hydroxybenzoate or sorbic acid, and if need, can contain conventional flavouring agent or coloring agent.
For injection, the liquid unit dosage forms of preparation contains active substance of the present invention and sterile carrier.According to carrier and concentration, this compound can be suspended or dissolve.The preparation of solution is normally by active substance being dissolved in a kind of carrier, and filter-sterilized before being packed into a kind of suitable bottle or ampoule, then seals.Adjuvant for example a kind of local anesthetic, antiseptic and buffer agent also can be dissolved in this carrier.In order to improve its stability, can be after packing bottle into, this compositions is freezing, and under vacuum, water is removed.
Pharmaceutical composition of the present invention, when being prepared into medicament, optionally add applicable medicine acceptable carrier, described medicine acceptable carrier is selected from: mannitol, sorbitol, sodium pyrosulfite, sodium sulfite, sodium thiosulfate, cysteine hydrochloride, TGA, methionine, vitamin C, EDETATE SODIUM, Ethylenediaminetetraacetic Acid Calcium Salt, the alkali-metal carbonate of monovalence, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulphuric acid, phosphoric acid, aminoacid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, cellulose and derivant thereof, alginate, gelatin, polyvinylpyrrolidone, glycerol, POLYSORBATE 80, agar, calcium carbonate, calcium bicarbonate, surfactant, Polyethylene Glycol, cyclodextrin, beta-schardinger dextrin-, phospholipid material, Kaolin, Pulvis Talci, calcium stearate, magnesium stearate etc.
Medical material plant of the present invention is widely distributed, and the extract that we set up simultaneously and the mature preparation process of monomer are stable, quality controllable reliable.
Accompanying drawing explanation
Fig. 1 a: the high aggressivity breast carcinoma of the people of take MDA-MB-231 is cell model, proves that stellera chamaejasme L extract is within the scope of low dosage, has very faint in vitro toxicity, does not have significant difference to the growth of cell and propagation impact.
Fig. 1 b: the high aggressivity breast carcinoma of the mice of take 4T1 is cell model, proves that stellera chamaejasme L extract is within the scope of low dosage, has very faint in vitro toxicity, does not have significant difference to the growth of cell and propagation impact.Take high aggressivity breast carcinoma 4T1, MDA-231 is model, take MTT experiment as detection means, result show low dosage stellera chamaejasme L extract and monomer component comparatively faint on the impact of the existence of cell and multiplication capacity, point out within the scope of this dosage place what safe dose.
Fig. 2: the high aggressivity breast carcinoma 4T1 of take is model, the Transwell of take experiment and Matrigel experiment are detection means, result shows the obviously motion Invasion Potential of inhibition tumor cell of low dosage stellera chamaejasme L extract and monomer component ICJ.
Fig. 3: in vivo test research shows, low dosage ICJ is comparatively faint on Mouse Weight impact, and no difference of science of statistics points out it to locate in vivo that what is nontoxic, low toxicity dosage range, and safety can ensure.
Fig. 4 a: the high aggressivity breast carcinoma of mice of luciferase labelling of take is disease model, carries out the original position mammary gland lotus tumor of mice and carries out pharmaceutical intervention.In vivo test research shows, low dosage ICJ can partly suppress the growth of mice original position breast carcinoma.In figure, small animal imaging photo is taken from after mice administration 25 days.
Fig. 4 b: after tumor-bearing mice tumor in situ excision, the volume of mice tumor in situ statistics.
Fig. 4 c: after tumor-bearing mice tumor in situ excision, the statistics that mice tumor in situ tumor is heavy.
Fig. 4 d: after tumor-bearing mice administration 25 days, the living together of the total number of photons of mice tumor in situ.
Fig. 5 a: in vivo test research shows, low dosage ICJ can clearly suppress the transfer of breast carcinoma tumor-bearing mice,
Small animal imaging photo is taken from latter 25 days of operation.
Fig. 5 b: it is abscissa that curve chart be take mice hands POD, the total number of photons of imaging of mice different number of days of take is vertical coordinate.
Fig. 6: utilize above-mentioned zooperal mice serum, by the method for ELISA, detect the impact of different pharmaceutical intervention on TGF-beta expression and secretion level in Mice Body.Result shows, ICJ can be within the scope of low dosage the level of TGF-beta in dose-dependent inhibition Mice Body.
Fig. 7: Western blot experimental result shows, take MDA-231 as cell model, low dosage ICJ processes expression that can dose-dependent rise CDH1, suppresses the expression of VIM, and then discloses its Effective Regulation effect to tumor cell EMT process from molecular level.
Fig. 8 a: confocal laser scanning microscope is tested and shown, low dosage ICJ processes, and can effectively reduce F-actin at intracellular tissue, reduces the polymerization of its fibers form.Reverse the polymerisation induced effect of TGF-beta to F-actin.
Fig. 8 b: confocal laser scanning microscope experiment shows, low dosage ICJ processing, the film location that can effectively induce CDH1, reverses film obscission and intracytoplasmic gathering that TGF-beta causes.
Fig. 9 a:Transwell experimental result shows, ICJ can reverse the activation of TGF-beta to MDA-231 cell movement ability from functional level.Clearly suppress the regulation and control impact of EMT process on cell movement ability.
The quantificational expression of Fig. 9 b:Transwell result.
Figure 10: Western Blot experimental result shows, ICJ can reverse the non-classical path of TGF-beta from molecular level, and each mainly participates in member's expression and phosphorylation level.The clear and definite inhibitory action of proof ICJ to the non-classical path of TGF-beta.Disclose it by suppressing non-classical path, inhibition TGF-beta expresses and the activation to EMT process, further pharmacological Mechanism and the behavior of inhibition tumor cell motion-invasion and attack-transfer.
The specific embodiment
By following specific embodiment, the present invention is further illustrated, but not as limitation of the present invention.
The preparation method of embodiment 1, stellera chamaejasme L extract
In the present embodiment, Stellera chamaejasme L. medical material 95% ethanol extraction of take is raw material, and first step normal phase column chromatography adopts
200～300 order silica gel, second step reversed phase column chromatography adopts 40～50 μ m ODS, the 3rd step high performance liquid chromatography preparation adopts 5 μ m C18 Bonded Phase preparative columns, and adopting the methanol-water solution of volumetric concentration 75% is eluent system, through three step separation, obtains 6 kinds of pure compounds.Concrete technology step is as follows:
(1) purification on normal-phase silica gel column chromatography: get Stellera chamaejasme L. medical material 95% ethanol extraction extractum, dissolve with one or both the mixed liquor in chloroform, methanol, under constantly stirring, add the 00 order column chromatography silica gel with the quality such as extractum, mix sample, fully dry under room temperature; Dry method loading afterwards, normal pressure descended 200～300 order 200～3 silicagel columns, successively with chloroform, chloroform-methanol 100:1, chloroform-methanol 50:1, chloroform-methanol 20:1 eluting, collected chloroform-methanol 20:1 stream part, after negative pressure concentration and recovery solvent, obtain one-level rough segmentation thing.
(2) anti-phase ODS column chromatography: by above-mentioned one-level rough segmentation thing with dissolve with methanol after, adding with the particle diameter of the quality such as one-level rough segmentation thing is that 40～50 μ m ODS column chromatography fillers are mixed sample, fully dries and be processed into powdery under room temperature;
Dry method loading afterwards, cross 40～50 μ m ODS chromatographic column (column length 40cm, internal diameter 8cm), medium lift pump pressurization, pressure is 5～10MPa, flow-control is at 50～100ml/min, successively with 15%, 30%, 45%, 60%, 70% methanol-water eluting, collect 70% methanol-water stream part, after negative pressure concentration and recovery solvent, obtain secondary rough segmentation thing.
The preparation of embodiment 2, extraction components monomeric compound and purification;
High performance liquid chromatography preparation: above-mentioned secondary rough segmentation thing is dissolved with DMSO, be mixed with the need testing solution that concentration is 70mg/ml, through 0.45 μ m filtering with microporous membrane; Chromatographic column is Chromatorex C18 preparative column, particle diameter 5 μ m, size 500 * 60mm; Sampling volume is 1.4ml, take volumetric concentration as 70% methanol-water solution be eluent system, flow speed control is at 60ml/min, online ultraviolet detection, detection wavelength set is 295nm, collects respectively stream part of containing compound 1,2,3,4,5,6, and negative pressure drying obtains 6 kinds of light brown powders, being purity is greater than 98% compound 1,2,3,4,5,6 sterlings (quality is 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 of above-mentioned 6 kinds of compounds and Structural Identification result 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 is C19H12O7.This compound identification is Isodaphnoretin.
Compound 2, light brown powder.UV（MeOH，）λmaxnm：215，295。CD(c=0.1mg/ml, MeOH) do not go out peak.MS（+ESI）m/z：557.148（[M+H]+）。Molecular weight 556, molecular formula is C31H24O10.This compound is new isomer, called after sikokianin D.
Compound 3, light brown powder.UV（MeOH，）λmaxnm：215，295。CD(c=0.1mg/ml, MeOH) do not go out peak.MS（+ESI）m/z：557.148（[M+H]+）。Molecular weight 556, molecular formula is C31H24O10.This compound identification is sikokianin 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 is C32H26O10.This compound identification is the peaceful B of (+)-Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae).
Compound 5, light brown powder.UV（MeOH，）λmaxnm：215，295。CD(c=0.2mg/ml, MeOH) do not go out peak.MS（+ESI）m/z：571.165（[M+H]+）。Molecular weight 570, molecular formula is C32H26O10.This compound identification is the peaceful B of the different Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae).
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 is C32H26O10.This compound is new isomer, the peaceful E of the called after Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae).
Inside and outside Pharmacodynamics screening and the evaluation of embodiment 3, Stellera chamaejasme L. extraction components and monomer component
Stellera chamaejasme L extract described in experiment is secondary rough segmentation thing in embodiment 1.
The monomer component of Stellera chamaejasme L. described in experiment (ICJ) is the peaceful B of (+)-Radix Euphorbiae Fischerianae (Radix Euphorbiae Ebracteolatae).
1 to take high aggressivity breast carcinoma be disease model, verified that Stellera chamaejasme L. is interval to the safe dose of the external existence of tumor cell and toxicity dose, for the stellera chamaejasme L extract research of low toxicity, safety is produced evidence by mtt assay.
Choose the high aggressivity breast carcinoma of people MDA-MB-231 cell and the high aggressivity breast carcinoma of mice 4T1 cell is cell model.Density with 3000 cells/well spreads into 96 orifice plates, and with bed board after within 24 hours, carry out drug treating, use variable concentrations stellera chamaejasme L extract to process tumor cell (ESC1ug/ml and ICJ0.5ug/ml), and in different time points harvesting (after drug treating 24/48/72 hour), by mtt assay, detect existence, the propagation level (in OD570 value and the cells survival rate of cell) of cell.
Experimentation shows, the stellera chamaejasme L extract of low dosage is processed, and can not obviously reduce the absorbance numerical value of drug treating group tumor cell, and the impact of tumor cell existence and multiplication capacity does not have significant difference.Therefore this stellera chamaejasme L extract that experimental results show that external low dosage does not have clear and definite drug toxicity, dosage safety is reliable.
2 to take high aggressivity breast carcinoma be disease model, by Transwell test, Matrigel experiment, scratch removal test, proof low dosage stellera chamaejasme L extract can be front lower in the prerequisite that does not affect cell proliferation and existence, effectively motion, the invasive ability of inhibition tumor cell.
In Transwell experiment, choose the high aggressivity breast carcinoma of people MDA-MB-231 cell and the high aggressivity breast carcinoma of mice 4T1 cell is cell model.Cell number by cell with 1*105 spreads in advance into 12 orifice plates, spreads latter 24 hours and in exponential phase, carries out drug treating, and system for handling is selected serum-free medium.Extraction components mixture medicines concentration for the treatment of is set as 1ug/ml, and monomeric compound drug treating concentration is set as 0.5ug/ml.After drug treating 24 hours, peptic cell is organized cell with the density of 50000/ cells/well and is spread in 24 hole transwell cells each.Transwell upper strata cell cultivating system is serum-free medium, and lower floor's cell cultivating system is 10% blood serum medium.Spread after cell, in cell culture incubator, cultivate 18 hours.After cultivation finishes, results, fixed cell, utilize swab stick softly to remove upper strata and do not wear theca cell, and use violet staining lower floor to wear theca cell.After dyeing, choose at random five high power lens visuals field, to wearing theca cell number, count, and each group count results is analyzed.
In Matrigel experiment, choose equally high aggressivity breast carcinoma MDA-MB-231 cell and the high aggressivity breast carcinoma of mice 4T1 cell is cell model.Progenitor cells processing and drug treating are with Transwell experimental design.In the cell of Transwell cell upper strata, prior to 4 ℃, spread into Matrigel glue (to specifications in advance, use culture medium 1:1 dilution colloid), and with 37 ℃ hatch 6 hours, make gel coagulation, and after coagulation, spread into drug treating and respectively organize cell, spreading into density is 80000/ hole, continues in cell culture incubator and cultivate 24 hours after spreading.Results, fixing, colouring method and result acquisition methods are with above-mentioned Transwell experimental technique.
By Transwell test, Matrigel experiments experiment proves, low dosage stellera chamaejasme L extract and monomer whose composition processed group, wear theca cell number and obviously reduce, and with negative control group, compares, and has clear and definite significant difference.Low dosage stellera chamaejasme L extract and monomer component be motion and the migration potential of inhibition tumor cell significantly.
3 to take the high aggressivity breast carcinoma of mice 4T1 be elementary cell model, builds the 4T1 cell strain with luciferase assay.And utilize this cell construction bearing mouse model, and by the method for small animal imaging, dynamic evaluation low dosage Stellera chamaejasme L. monomer component is grown on tumor cancer in situ in vivo impact and the inhibition to metastatic tumour thereof.Experiment shows that low dosage Stellera chamaejasme L. monomer component (ICJ) can be in not affecting the low toxicity dosage range of Mouse Weight, part suppresses the growth of mice tumor in situ, obviously suppress the transfer of mouse breast cancer simultaneously, this result has been supported the pharmacodynamics evidence of experiment in vitro, from level in body, provides Research foundation for its antimetastatic activity.
In vivo in pharmacodynamic evaluation, modeling method is inoculated in the 4th pair of breast pad of BALB/C mice for the density with 10000/Mus.In model construction, after one week, carry out small animal imaging detection (100ul fluorescein potassium salt/Mus), by the number of total number of photons, mice is carried out to random packet, guarantee that each experimental group and the total number of photons of matched group small mouse do not possess significant difference.After grouping, by the method for lumbar injection, according to the drug level of 0ug/kg30ug/kg300ug/kg, mice is carried out intervening in medicine body, inject once every day, and each drug injection system is 100ul/ Mus.Pharmaceutical intervention is carried out 36 days continuously, during every three days, carry out small animal imaging, the growing state of dynamic, noninvasive monitoring tumor-bearing mice tumor in situ.After administration 36 days, by excision mice tumor in situ, calculate the volume and weight of tumor in situ, observe transfering state and the intensity (in total number of photons) of original position breast carcinoma simultaneously, and according to former drug level and medication, continue administration 25 days after primary tumor excision, pass judgment on the inhibition of drug exposure to metastatic tumour.After 25 days, put to death mice, the internal organs such as results mouse lung, liver, in order to subsequent examination.
Experimental result shows, low dosage ICJ is comparatively faint on Mouse Weight impact, respectively gives medicine with negative control group Mouse Weight no difference of science of statistics.The toxicity in vivo of proof low dosage ICJ is comparatively faint.In low toxicity dosage range, ICJ can partly suppress the volume and weight of tumor in situ in vivo, and part suppresses total number of photons of tumor in situ.Meanwhile, ICJ can be within the scope of low dosage metastasis number and the transition intensity (in total number of photons) of dose-dependent inhibition metastatic tumor.Therefore, in body, experiment clearly proves that low toxicity ICJ can partly suppress the growth of tumor in situ, obviously suppresses the transfer process of tumor, has effective and clear and definite anti metastasis drug effect.The pharmacological mechanism research of embodiment 4, low dosage stellera chamaejasme L extract and monomer component
1 utilizes the animal experimental model in last minute, obtains and respectively organizes laboratory animal serum, utilizes ELISA method, the expression of TGF-beta in detection of drugs processed group blood serum sample, and analyze the affect trend of drug treating on TGF-beta.Experiment showed, that stellera chamaejasme L extract and monomer component can effectively suppress the expression of TGF-beta in the interior serum of tumor-bearing mice body.
By plucking eyeball, get the method for blood, from the laboratory animal of second portion the 3rd title, obtain each drug treating group and negative control group animal serum.And appropriateness dilution respectively organizes serum (dilution ratio is 1:30), utilize this blood serum sample to detect by ELISA method.ELISA testing result shows, each low dosage Stellera chamaejasme L. group is processed TGF-beta content in animal serum and is obviously reduced, therefore originally experiment showed, low dosage stellera chamaejasme L extract and the monomer component expression and secretion of dose-dependent inhibition TGF-beta in vivo.
2 to take high aggressivity breast carcinoma MDA-231 and 4LK cell be model, by WesternBlot, detect the expression that proof low dosage stellera chamaejasme L extract can obviously promote antioncogene and epithelium molecule core mark CDH1, suppress the expression of interstitial core molecular marker VIM, the EMT process of modulate tumor cell, between inhibition tumor cell, materialization changes, and recovers the epithelium attribute of tumor cell from molecular level.
Cell spreads into 6 orifice plates with the number of 2*105, and in spreading within latter 24 hours, carrying out pharmaceutical intervention, drug treating concentration is set as 0.1/0.5/1ug/ml ICJ, continue to cultivate 24/48 hour, and in each time point harvesting, utilize RIPA lysate to extract total cellular protein extract, and utilize Westernblot technology, detect expression and the phosphorylation level of associated protein in each drug treating group.
3 to take breast carcinoma MDA-231 and ZR75-1 cell be model, by IIF method, detects the impact of Stellera chamaejasme L. monomer component ICJ on CDH1 in tumor cell and F-actin Subcellular Localization and form.And evaluate its blocking effect to TGF-beta.
Utilize 20ng/mlTGF-beta pretreatment tumor cell 24 hours, and with process after add low dosage Stellera chamaejasme L. monomer component ICJ (1ug/ml), continue drug effect 48 hours, in corresponding time point harvesting, by IIF method, detect the distribution situation of CDH1 in cell: first antibody is selected CDH1 antibody, antibody dilution ratio is 1:200, and second antibody selects the rabbit two of FITC labelling anti-, and dilution ratio is 1:200.And in the successfully laggard row DAPI dyeing of dyeing, stin of thickness is 100ng/ml.After dyeing, carry out confocal laser scanning microscope and take pictures.
4 to take breast carcinoma MDA-231 cell be model, by Transwell, test, proof low dosage stellera chamaejasme L extract can obviously be blocked the enhancing of the Tumor Cell Migration ability of TGF-beta induction, maintain tumor cell under TGF-beta induction motion transfer ability at reduced levels, thereby from functional level antagonism TGF-beta the facilitation to Tumor Cell Migration-transfer ability.Thereby the drug target molecule of proof ICJ is TGF-beta.
Utilize 20ng/mlTGF-beta pretreatment tumor cell 36 hours, and with process after add low dosage Stellera chamaejasme L. monomer component ICJ (1ug/ml), continue drug effect 24/48 hour, in each time point harvesting, utilize RIPA lysate to extract total cellular protein extract, and utilize Westernblot technology, detect expression and the phosphorylation level of associated protein in each drug treating group.
5 to take breast carcinoma MDA-231 cell and ZR75-1 cell be model, by WesternBlot, detect proof: low dosage stellera chamaejasme L extract can obviously reverse the tumor cell EMT process that suppresses TGR-beta induction, remain on the expression of the tumor cell epithelium molecule core mark (CDH1 etc.) under TGF-beta induction, suppress the up-regulated of the interstitial core molecular marker (VIM etc.) of TGF-beta induction.Thereby from molecular level, prove, ICJ can induction and the impact of antagonism TGF-beta on tumor cell EMT process.Thereby the drug target molecule of further clear and definite ICJ is TGF-beta.
Utilize 20ng/mlTGF-beta pretreatment tumor cell 36 hours, and with process after add low dosage Stellera chamaejasme L. monomer component ICJ (1ug/ml), continue drug effect 24/48 hour, in each time point harvesting, utilize RIPA lysate to extract total cellular protein extract, and utilize Westernblot technology, detect expression and the phosphorylation level of associated protein in each drug treating group.
6 to take breast carcinoma MDA-231 cell be model, crosses WesternBlot and detect, the activation levels of checking TGF-beta coherent signal path.Experimental result shows, ICJ processes after tumor cell, in the non-classical path of TGF-beta, expression (ITGB3) phosphorylation level (FAK/p38) of each important composition molecule all shows clear and definite inhibitory state, thereby from signal path level, further clear and definite Stellera chamaejasme L. monomer component ICJ can be take TGF-beta as drug target, by suppressing the activation of the non-classical signal path of TGF-beta, motion-migration the level that has further suppressed tumor cell, and then final, suppressed invasion and attack and the transfer of tumor.
Utilize 20ng/mlTGF-beta pretreatment tumor cell 36 hours, and with process after add low dosage Stellera chamaejasme L. monomer component ICJ (1ug/ml), continue drug effect 24/48 hour, in each time point harvesting, utilize RIPA lysate to extract total cellular protein extract, and utilize Westernblot technology, detect expression and the phosphorylation level of associated protein in each drug treating group.
Claims (10)
1. Stellera chamaejasme L. or its extract or the monomer component application in the medicine of preparation anti metastasis.
2. application claimed in claim 1, is characterized in that, Stellera chamaejasme L. or its extract or monomer component have the function balance that regulates TGF-beta, promotes the drug activity of the Remolding of Functions of TGF-beta in tumor cell.
3. application claimed in claim 1, it is characterized in that, Stellera chamaejasme L. or its extract or monomer component have the expression that promotes antioncogene and epithelium molecule core mark CDH1, suppress the expression of interstitial core molecular marker VIM, the EMT process of modulate tumor cell, between inhibition tumor cell, materialization changes, and recovers the effect of the epithelium attribute of tumor cell from molecular level.
4. application claimed in claim 1, it is characterized in that, Stellera chamaejasme L. or its extract or monomer component have the Tumor Cell Migration ability of blocking-up TGF-beta induction and the enhancing of invasive ability, maintain tumor cell under TGF-beta induction migration-invasive ability at reduced levels, thereby the facilitation from functional level antagonism TGF-beta to tumor cell migration-invasive ability, meanwhile, level, can obviously suppress the high transition intensity that shifts strain of breast carcinoma in vivo.
5. application claimed in claim 1, it is characterized in that, Stellera chamaejasme L. or its extract or monomer component have the tumor cell EMT process that suppresses TGR-beta induction that reverses, remain on the expression of the tumor cell epithelium molecule core mark CDH1 under TGF-beta induction, the up-regulated that suppresses the interstitial core molecular marker VIM of TGF-beta induction, thereby induction and the impact of antagonism TGF-beta on tumor cell EMT process.
6. application claimed in claim 1, is characterized in that, described stellera chamaejasme L extract or monomer component both can buy on market, also can obtain by the processing of routine techniques means.
7. the application of the pharmaceutical composition that contains Stellera chamaejasme L. or its extract or monomer component in the medicine of preparation anti metastasis.
8. application claimed in claim 7, is characterized in that, also contains pharmaceutically acceptable carrier in pharmaceutical composition.
9. application claimed in claim 7, is characterized in that, pharmaceutical composition can be prepared into any pharmaceutically acceptable dosage form.
10. application claimed in claim 9, it is characterized in that, pharmaceutical dosage form comprises: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, suck agent, granule, electuary, pill, powder, unguentum, sublimed preparation, suspensoid, powder, solution, injection, suppository, ointment, plaster, cream, spray, drop, patch.Preparation of the present invention, peroral dosage form preferably, as: capsule, tablet, oral liquid, granule, pill, powder, sublimed preparation, unguentum etc.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1554421A (en) * | 2003-12-23 | 2004-12-15 | 泽 王 | Tumour control pill |
| CN103405721A (en) * | 2013-08-22 | 2013-11-27 | 张素平 | Pharmaceutical composition for inhibiting tumor metastasis and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1554421A (en) * | 2003-12-23 | 2004-12-15 | 泽 王 | Tumour control pill |
| CN103405721A (en) * | 2013-08-22 | 2013-11-27 | 张素平 | Pharmaceutical composition for inhibiting tumor metastasis and application thereof |
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| 冯宝民等: "瑞香狼毒中的化学成分", 《中草药》 * |
| 阚晓溪等: "瑞香狼毒醇提物的抗肿瘤活性研究", 《中国中药杂志》 * |
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