CN105477020A - Anti-glioma drugs based on Venenum Bufonis extract and preparation method thereof - Google Patents
Anti-glioma drugs based on Venenum Bufonis extract and preparation method thereof Download PDFInfo
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Abstract
The invention relates to antitumor drugs and discloses anti-glioma drugs based on Venenum Bufonis extract and a preparation method thereof, the drugs being a bufotalin submicron emulsion and a bufotalin nanoparticle preparation, wherein the bufotalin submicron emulsion comprises Venenum Bufonis extract, medium chain triglyceride, a lecithin, poloxamer 188, glycerol, sodium oleate and injection water; the bufotalin nanoparticle preparation comprises Venenum Bufonis extract, glycerol monostearate, medium-chain fatty acid glyceride, oleic acid, a lecithin, poloxamer 188, sodium deoxycholate and injection water. Both the two preparations can combine with endothelial cells in cerebral blood capillaries, enabling the drugs to be transmitted into the brain through membranes or by means of adsorption, medication, endocytosis and transferring, cerebral targeting of the drugs is improved, and further increase of bufotalin in the brain is facilitated, and glioma resistance is achieved by inducing apoptosis and excessive autophagy of cells.
Description
Technical field
The present invention relates to antitumor drug, particularly based on the anti-cerebral glioma medicine and preparation method thereof of Venenum Bufonis extract.
Background technology
Cerebral glioma is the modal constitutional intracranial tumour of one because brain and the canceration of spinal cord glial cell produce, and accounts for 35% ~ 61% of intracranial tumor, and its aggressive is strong, and poor prognosis is the malignant tumor that case fatality rate and disability rate are all higher.Cerebral glioma, at the beginning of morbidity, usually do not have typical symptom, and glioma is positioned at the invasive growth feature in critical function district and deep, makes it be difficult to entirely cut, also very inresponsive to chemicotherapy, is very easy to recurrence.And chemicals and general anti-tumor Chinese medicine, kill and wound brain glioblastoma cell and through blood brain barrier two aspect effect, curative effect is also undesirable because being difficult to have simultaneously, cause cerebral glioma to be still one of tumor that in general tumour, prognosis is the poorest so far.Therefore, the novel anti-cerebral glioma chemotherapeutics of research and development relative efficiency low toxicity becomes problem demanding prompt solution clinically, is also advanced subject and the hot fields of our times research simultaneously.
Summary of the invention
For problems of the prior art, the object of the present invention is to provide the anti-cerebral glioma medicine and preparation method thereof based on Venenum Bufonis extract, said preparation can be combined by the endotheliocyte in cerebral capillary, medicine permeable membrane is transmitted into brain, or gulp down transhipment effect by absorption mediation bag, improve the brain targeting of medicine, thus the distribution of bufotalin in brain can be made to increase further, played the effect of anti-cerebral glioma by the apoptosis of inducing cell.
In order to achieve the above object, the present invention is achieved by the following technical programs.
Technical scheme one:
A kind of anti-cerebral glioma medicine based on Venenum Bufonis extract of the present invention, for bufotalin submicron emulsion preparation (BU-SCP), it is characterized in that, its 100mL standard dose component is: 50mg Venenum Bufonis extract, 10g medium chain triglyceride (MCT), 3g lecithin, 0.2g PLURONICS F87 (F-68), 2.5g glycerol, 0.05g enuatrol, all the other are water for injection.
In described Venenum Bufonis extract, the comprehensive content of Toadpoison Medicine, cinobufagin and bufogenin is greater than 95%.
The preparation method of above-mentioned bufotalin submicron emulsion preparation (BU-SCP), it is characterized in that, the preparation of its 100mL standard dose comprises the following steps:
(1) by 3g lecithin and 50mg Venenum Bufonis extract, add in 10g medium chain triglyceride (MCT) respectively, heated and stirred is dissolved, and obtains the oil phase of the pastille clarified;
(2) add in water for injection by 0.2g PLURONICS F87 (F-68), 0.05g enuatrol and 2.5g glycerol, at 70 DEG C, heated and stirred makes dissolving obtain aqueous phase;
(3) under the stirring of high speed dispersor, oil phase is added in aqueous phase, continues to stir, obtained colostrum;
(4) colostrum is used hydrochloric acid solution adjust ph, be settled to 100mL with water for injection, cooling, is transferred to homogenizing in high pressure homogenizer, bottling, inflated with nitrogen, sealing, autoclaving, in psychrolusia cooling after taking-up, to obtain final product.
The further improvement of technique scheme and feature are:
In step (1), the temperature of described heating is 78 DEG C, and the time is 30min.
In step (3), the rotating speed of described high speed dispersor is 12,000rpm, and mixing time is 3min.
In step (4), the concentration of described hydrochloric acid solution is 0.1molL
-1, pH value is between 5 ~ 6.5.
In step (4), the pressure of high pressure homogenizer is 800bar, homogenizing 8 times.
In step (4), autoclave temperature is 121 DEG C, and the time is 10min.
Technical scheme two:
A kind of anti-cerebral glioma medicine based on Venenum Bufonis extract of the present invention, for bufotalin nano particle preparations (BU-NPP), it is characterized in that, its 100mL standard dose component is: 50mg Venenum Bufonis extract, 2.0 glyceryl monostearate, 0.8g medium chain length fatty acid triglyceride, 0.5g oleic acid, 2.0g lecithin (Lipoid
), 1.5g PLURONICS F87 (PluronicF68), 0.3g NaTDC, all the other are water for injection.
In described Venenum Bufonis extract, the comprehensive content of Toadpoison Medicine, cinobufagin and bufogenin is greater than 95%.
The preparation method of above-mentioned bufotalin nanoparticle (BU-NPP), is characterized in that, comprise the following steps:
(1) take 50mg Venenum Bufonis extract, 2.0g glyceryl monostearate, 0.8g medium chain length fatty acid triglyceride, 0.5g oleic acid, heating in water bath lower magnetic force stirring and dissolving, obtain transparent homogeneous oil phase;
(2) by 2.0g lecithin (Lipoid
), 1.5g PLURONICS F87 (PluronicF68), 0.3g NaTDC is added in water for injection, and heat under magnetic agitation, dispersing and dissolving obtains aqueous phase;
(3) under magnetic stirring, be added dropwise to by aqueous phase while hot in identical temperature oil phase, ultrasonic disperse, under stirring condition, ice-water bath cools, and under aseptic condition, uses filter membrane Entkeimung, to obtain final product.
The further improvement of technique scheme and feature are:
In step (1), bath temperature is 75 DEG C.
In step (2), the rotating speed of described magnetic agitation is 500r/min, and heating-up temperature is 75 DEG C.
In step (3), described aqueous phase rate of addition is 10mL/min, and the magnetic agitation time is 5min, and ultrasonic time is 10min, and filter sizes is 0.22 μm.
The application of the present invention in the anti-cerebral glioma medicine of preparation, is detected by the glioma U87-MG cell MTT of BU-S and the BU-SCP process to variable concentrations, proves the lethal effect to glioma cell; BU-SCP is detected on apoptotic impact in conjunction with flow cytometry (FCM) by the two transfection reagent box of AnnexinV-FITC/PI; By the BU-SCP of variable concentrations, Orthotopic implantation in nude mice cerebral glioma is acted on, with routine clinical medicine temozolomide injection (Temozolomide, TMZM) and negative control group compare, detect the volume of nude mice cerebral glioma and the signal intensity of tumor by Imaging-PAM in nuclear magnetic resonance scanning technology and living animal body; By HE dyeing (hematoxylin-eosin staining method, hematoxylin-eosinstaining, HE), pathological examination is carried out to cerebral glioma; The expression of immunohistochemistry technique inspection (S-100) and (GFAP) albumen; Prove with this, BU-S and BU-SCP has the effect of anti-cerebral glioma in body, and BU-S with BU-SCP is relevant with the apoptosis of inducing cell to the lethal effect of cerebral glioma.
Compared with prior art, the present invention has the following advantages and useful technique effect:
(1) outward appearance of bufotalin submicron emulsion preparation (BU-SCP) that prepared by the present invention is white uniformity emulsion liquid, and particle diameter 100 ~ 200nm, zeta current potential-30 ~ 40mV, the envelop rate of medicine is more than 85%, and pH value is 5 ~ 6.5.
(2) the bufotalin nanoparticle (BU-NPP) that prepared by the present invention is the clear liquid with blue-opalescent, and particle diameter 50 ~ 100nm, zeta current potential is at-15 ~ 20Mv, and the envelop rate of medicine is more than 85%, and pH value is 6.5 ~ 7.5.
(3) MTT detection cell viability shows, the cell inhibitory effect of BU-SCP and BU-NPP has obvious dose-effect relationship, AnnexinV-FITC/PI flow cytometer result shows, along with the increase of BU-SCP and BU-NPP concentration, the apoptosis rate of U87-MG cell obviously rises; The amynologic label albumen (S-100) of Showed by immune group result brain tumor diagnosis and the specific expressed albumen (GFAP) of Astrocytic glioma, respectively at endochylema and karyon high expressed, show nude mice modeling success; The signal intensity that in living animal body, fluorescence imaging detects each group of nude mice in-vivo tumour shows that BU-SCP and BU-NPP plays offect of ntiglioma in nude mouse, in the scope of data of experiment, become dose dependent, and the offect of ntiglioma of BU-SCP and BU-NPP is strong; The volume of nuclear magnetic scanning Technique dynamic monitoring nude mice cerebral glioma shows that the inhibited proliferation of BU-SCP with BU-NPP to nude mice cerebral glioma becomes dose dependent.M-ly when BU-SCP and BU-NPP is dose-dependently significantly suppress U87-MG Growth of Cells, by the apoptosis killing tumor cell of inducing cell, BU-SCP and BU-NPP all has obvious anti-cerebral glioma active in nude mouse, and BU-SCP and BU-NPP is dose dependent in scope of experiment.
(4) bufotalin submicron emulsion preparation (BU-SCP) and bufotalin nanoparticle (BU-NPP) can be combined by the endotheliocyte in cerebral capillary, medicine permeable membrane is transmitted into brain, or gulp down transhipment effect by absorption mediation bag, improve the brain targeting of medicine, thus the distribution of bufotalin in brain can be made to increase further, the effect of anti-cerebral glioma is played by the apoptosis of inducing cell.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is described in further details.
Fig. 1 is the schematic diagram of BU-S to the effect of human glioma U87-MG cell inhibitory effect, in figure, abscissa is the drug level of BU-S, vertical coordinate is the suppression ratio of U87-MG Growth of Cells, and three Trendline in figure are followed successively by the growth inhibition ratio of cell after variable concentrations drug effect 24h, 48h and 72h from the bottom up.
Fig. 2 is the schematic diagram of BU-SCP of the present invention to the effect of human glioma U87-MG cell inhibitory effect, in figure, abscissa is the drug level of BU-SCP, vertical coordinate is the suppression ratio of U87-MG Growth of Cells, and three Trendline in two figure are followed successively by the growth inhibition ratio of cell after variable concentrations drug effect 24h, 48h and 72h from the bottom up.
Fig. 3 be BU-SCP of the present invention on the scatterplot of the apoptotic impact of U87-MG at bivariate flow cytometer, wherein, abscissa is AnnexinV, vertical coordinate is PI, left lower quadrant is living cells, and right lower quadrant is the early stage cell of apoptosis, and right upper quadrant is apoptosis late cell.Control is Normal group, and BU-SCPlow, BU-SCPmiddle and BU-SCPhigh are respectively three dosage groups (10,30 and 60 μ g/ml) of BU-SCP.
Fig. 4 is the body weight change comparison diagram after different dosing group nude mice of the present invention treatment, in figure, abscissa is different dosing group, from left to right be followed successively by blank group, low middle high three dosage (0.05mg/ml, 0.1mg/ml and 0.2mg/ml) groups of BU-SCP, BU-S (0.1mg/ml) group and positive drug TMZM (2.5mg/ml) matched group, often group includes the body weight of nude mice before modeling, the body weight of 12 days nude mices after the body weight of 14 days nude mices and administration after modeling; Vertical coordinate represents the body weight change of different group nude mice.
Fig. 5 is band tumor comparison diagram life cycle of different dosing group nude mice of the present invention, in figure, abscissa is different dosing group, from left to right be followed successively by blank group, low middle high three dosage (0.05mg/ml, 0.1mg/ml and 0.2mg/ml) groups of BU-SCP, BU-S (0.1mg/ml) group and positive drug TMZM (2.5mg/ml) matched group, vertical coordinate represents the life span of different group nude mice band tumor.
Fig. 6 is that fluorescence imaging of the present invention detects tumor signal intensity map in different dosing group nude mouse, in figure, abscissa is different dosing group, from left to right be followed successively by blank group, low middle high three dosage (0.05mg/ml, 0.1mg/ml and 0.2mg/ml) groups of BU-SCP, BU-S (0.1mg/ml) group and positive drug TMZM (2.5mg/ml) matched group, vertical coordinate represents tumor signal intensity in different group nude mouse.
Fig. 7 is the change in volume figure of different dosing group nude mice cerebral glioma of the present invention, in figure, abscissa is different dosing group, from left to right be followed successively by blank group, high three dosage (0.05mg/ml, 0.1mg/ml and 0.2mg/ml) groups at the bottom of BU-SCP, BU-S (0.1mg/ml) group and positive drug TMZM (2.5mg/ml) matched group, vertical coordinate represents the volume of tumor in different group nude mice brain.
Fig. 8 is the tumour inhibiting rate figure of different dosing group nude mice of the present invention, in figure, abscissa is different dosing group, from left to right be followed successively by blank group, low middle high three dosage (0.05mg/ml, 0.1mg/ml and 0.2mg/ml) groups of BU-SCP, BU-S (0.1mg/ml) group and positive drug TMZM (2.5mg/ml) matched group, vertical coordinate represents the tumour inhibiting rate of different group nude mice.
Fig. 9 is pathological examination (the HE dyeing of different dosing group cerebral glioma of the present invention, × 200) Normal group, model group, low middle high three dosage (0.05mg/ml, 0.1mg/ml and 0.2mg/ml) groups of BU-SCP, BU-S (0.1mg/ml) group and positive drug TMZM (2.5mg/ml) matched group is from left to right followed successively by figure, figure.
Figure 10 is the expression that in immunohistochemical staining (× 400) figure, the figure of cerebral glioma S-100 albumen, S-100 (-) is normal cerebral tissue S-100 albumen, and S-100 (+) is the expression of tumor tissues S-100 albumen.
Figure 11 is the expression that in immunohistochemical staining (× 400) figure, the figure of cerebral glioma GFAP albumen, GFAP (-) is normal cerebral tissue GFAP albumen, and GFAP (+) is the expression of tumor tissues GFAP albumen.
Detailed description of the invention
Below provide concrete experimental example so that the present invention is described in further detail, but the invention is not restricted to these experimental examples.
Experimental example 1:
Prepare the Venenum Bufonis extract that the comprehensive content of Toadpoison Medicine, cinobufagin and bufogenin is greater than 95%, comprise the following steps:
(1) Venenum Bufonis pulverizing medicinal materials is become coarse powder, too 60 mesh sieves, add in the ethanol of volumetric concentration 80% and dissolve, wherein, the quality of ethanol is 10 times of meal quality, and be dissolved as heating in water bath and dissolve, water bath heating temperature is 80 DEG C, and heat time heating time is 1h; Then sucking filtration, the ethanol of evaporation of filtrate, dry, obtain crude extract.
(2) crude extract is added distilled water, wherein the quality of distilled water is 10 times of the quality of crude extract, and ultrasound suspending, obtains suspension; Add extraction into ethyl acetate again, wherein 10 times of quality of the quality suspension of ethyl acetate, obtain extract; Ethyl acetate in evaporation liquid, and vacuum drying, be extracted thing.
(3) be dissolved in by extract in the chloroform that volume ratio is 1:1 and methyl alcohol mixed liquor, wherein the quality of chloroform and methyl alcohol mixed liquor is 1.5 times of the quality of extract, and elimination insoluble matter, obtains clear liquor; Stirred by clear liquor limit edged and mix sample in silica gel, wherein the quality of silica gel is 5 times of the quality of clear liquor, places and treats bone dry, carry out silica gel column chromatography separation.
When silica gel column chromatography is separated, be first the cyclohexane-acetone mixed liquor eluting 3 times of 10:1 by volume ratio, then be the cyclohexane-acetone mixed liquor eluting 5 times of 5:1 by volume ratio, then use acetone eluting 1 time, finally use methanol-eluted fractions 1 time, obtain mixing eluent.
(4) mixing eluent is carried out distilling under reduced pressure, evaporation eluant, and fraction is concentrated, dry, obtain Venenum Bufonis extract.
Adopt thin layer chromatography (TLC), measure Toadpoison Medicine, cinobufagin and bufogenin content in Venenum Bufonis extract, calculate the purity of Toadpoison Medicine, cinobufagin and bufogenin.In the Venenum Bufonis extract of the present embodiment, Toadpoison Medicine (Bufalin) content 20%, cinobufagin (Cinobufagin) content be 30% and bufogenin (Resibufogenin) content be 46%, the content 96% of three.
Venenum Bufonis anticancer effective component is fat-soluble bufotalin compounds mainly, wherein Toadpoison Medicine (Bufalin), cinobufagin (Cinobufagin) and bufogenin (Resibufogenin) are higher, active three the stronger compositions of content in bufotalin, are white or broken white powder.
Experimental example 2:
Regular solution agent (the bufadienolidessolution of above-mentioned Venenum Bufonis extract, BU-S), for anti-cerebral glioma medicine, its 5mL standard dose component is: 2.5mg Venenum Bufonis extract, 2.5mL first solvent propylene glycol, all the other are the second solvent injection water.Wherein, in described Venenum Bufonis extract, the comprehensive content of Toadpoison Medicine, cinobufagin and bufogenin is greater than 95%.The Venenum Bufonis extract that this experimental example 2 adopts experimental example 1 to prepare.
The preparation method of the regular solution agent of above-mentioned Venenum Bufonis extract, comprises the following steps: weighed Venenum Bufonis extract 2.5mg puts in 5mL measuring bottle, adds propylene glycol 2.5mL, ultrasonic dissolution, injects dilute with water standardize solution 5mL, shakes up, and filters, to obtain final product with 0.22 μm of filter membrane.
Experimental example 3:
A kind of anti-cerebral glioma medicine based on Venenum Bufonis extract of the present invention, for bufotalin submicron emulsion preparation (BU-SCP), its 100mL standard dose component is: 50mg Venenum Bufonis extract, 10g medium chain triglyceride (MCT), 3g lecithin, 0.2g PLURONICS F87 (F-68), 2.5g glycerol, 0.05g enuatrol, all the other are water for injection.Wherein, in described Venenum Bufonis extract, the comprehensive content of Toadpoison Medicine, cinobufagin and bufogenin is greater than 95%.The Venenum Bufonis extract that this experimental example 3 adopts experimental example 1 to prepare.
The preparation method of above-mentioned bufotalin submicron emulsion preparation (BU-SCP), it is characterized in that, the preparation of its 100mL standard dose comprises the following steps:
(1) by 3g lecithin and 50mg Venenum Bufonis extract, add in 10g medium chain triglyceride (MCT) respectively, heated and stirred is dissolved, and obtains the oil phase of the pastille clarified; Wherein, the temperature of heating is 78 DEG C, and the time is 30min.
(2) add in water for injection by 0.2g PLURONICS F87 (F-68), 0.05g enuatrol and 2.5g glycerol, at 70 DEG C, heated and stirred makes dissolving obtain aqueous phase.
(3) under the stirring of high speed dispersor, oil phase is added in aqueous phase, continues to stir, obtained colostrum; Wherein, the rotating speed of high speed dispersor is 12,000rpm, and mixing time is 3min.
(4) colostrum is used hydrochloric acid solution adjust ph, be settled to 100mL with water for injection, cooling, is transferred to homogenizing in high pressure homogenizer, bottling, inflated with nitrogen, sealing, autoclaving, in psychrolusia cooling after taking-up, to obtain final product.Wherein, the concentration of hydrochloric acid solution is 0.1molL
-1, pH value is between 5 ~ 6.5; The pressure of high pressure homogenizer is 800bar, homogenizing 8 times; Autoclave temperature is 121 DEG C, and the time is 10min.
Experimental example 4:
A kind of anti-cerebral glioma medicine based on Venenum Bufonis extract of the present invention, for bufotalin nano particle preparations (BU-NPP), its 100mL standard dose component is: 50mg Venenum Bufonis extract, 2.0 glyceryl monostearate, 0.8g medium chain length fatty acid triglyceride, 0.5g oleic acid, 2.0g lecithin (Lipoid
), 1.5g PLURONICS F87 (PluronicF68), 0.3g NaTDC, all the other are water for injection.Wherein, in Venenum Bufonis extract, the comprehensive content of Toadpoison Medicine, cinobufagin and bufogenin is greater than 95%.The Venenum Bufonis extract that this experimental example 4 adopts experimental example 1 to prepare.
The preparation method of above-mentioned bufotalin nano particle preparations (BU-NPP), comprises the following steps:
(1) take 50mg Venenum Bufonis extract, 2.0g glyceryl monostearate, 0.8g medium chain length fatty acid triglyceride, 0.5g oleic acid, heating in water bath lower magnetic force stirring and dissolving, obtain transparent homogeneous oil phase; Wherein, bath temperature is 75 DEG C.
(2) by 2.0g lecithin (Lipoid
), 1.5g PLURONICS F87 (PluronicF68), 0.3g NaTDC is added in water for injection, and heat under magnetic agitation, dispersing and dissolving obtains aqueous phase; Wherein, the rotating speed of described magnetic agitation is 500r/min, and heating-up temperature is 75 DEG C.
(3) under magnetic stirring, be added dropwise to by aqueous phase while hot in identical temperature oil phase, ultrasonic disperse, under stirring condition, ice-water bath cools, and under aseptic condition, uses filter membrane Entkeimung, to obtain final product.Wherein, aqueous phase rate of addition is 10mL/min, and the magnetic agitation time is 5min, and ultrasonic time is 10min, and filter sizes is 0.22 μm.
Experimental example 5:
The inhibitory action of BU-S and BU-SCP to human glioma cell U87-MG in-vitro multiplication is described.
Cell culture: U87-MG brain glioblastoma cell, containing in the DMEM culture fluid of volume ratio 10% hyclone, is placed in 37 DEG C, 5% volumetric concentration CO
2cultivate in incubator, when cell concentration grows to 80%-90% (exponential phase), with mass percent 0.25% trypsin and mass percent 0.02% ethylenediaminetetraacetic acid (ethylenediamineetraaceticacid, EDTA) digest, washing, resuspended, be diluted to 1x10
5the single cell suspension of individual/ml, be seeded in 96 orifice plates, be placed in after cultivating 24h in incubator, discard culture fluid, add the complete cell culture fluid of BU-SCP and BU-S (1.625 μ g/ml, 3.25 μ g/ml, 6.5 μ g/ml, 15 μ g/ml, 30 μ g/ml, 60 μ g/ml) containing variable concentrations, hatch altogether different time 24,48, after 72h, adopt Methyl thiazoly tetrazolium assay method (MTT) to measure cell viability.
Result as shown in Figure 1 and Figure 2, along with the increase of drug level and the prolongation of action time, also stronger to the growth inhibited effect of U87-MG cell, namely BU-S and BU-SCP is dosage and time dependence to the effect of U87-MG cell inhibitory effect, and the cell inhibitory effect effect of same concentrations BU-SCP is stronger than BU-S.
Experimental example 6:
Flow cytometry (Flowingcellmicmspectrofluorimetry, FCM) detects apoptotic cell.
Take the logarithm trophophase U87-MG cell, digestion, washing, resuspended, be seeded in 6 orifice plates, every hole inoculation 1ml is containing 1*10
6the complete cell culture fluid DMEM of individual cell.Cultivate 24h, discard culture fluid.Every hole adds containing variable concentrations BU-SCP (10 μ g/ml, 30 μ g/ml, 60 μ g/ml) again, after mixing, is placed in incubator and continues cultivation 24 hours with U87-MG cell.With mass percent 0.25% trypsin and mass percent 0.02% ethylenediaminetetraacetic acid (EDTA) peptic cell, add the complete medium cessation reaction containing serum; Slight piping and druming makes U87-MG cell take off wall, the centrifugal 5min collecting cell of 800rpm, supernatant discarded, with phosphate buffer (PBS) washed cell 2 times; Subsequent step operates according to AnnexinV-FITC test kit description, uses flow cytometric analysis Level of Apoptosis.
Result as shown in Figure 3, 10, 30, after 60ug/mlBU-SCP acts on U87-MG cell 24h, AnnexinV/PI analyzes each medication group apoptosis rate and is respectively (9.6% ± 1.1) %, (22.2% ± 1.7) %, (26.9% ± 2.4) %, each group of apoptosis rate is apparently higher than blank group (0.5% ± 0.02) the % same period, each administration group comparatively matched group has significant difference (P<0.01), illustrate that the apoptosis rate of each administration group raises with the increase of drug dose, BU-SCP is by the apoptosis killer cell of induction U87-MG cell.
Experimental example 7:
The effect experiment of the anti-cerebral glioma of BU-SCP and BU-S.
(1) cell culture: it is 75cm that human glioma U87-MG cell is sub-packed in several floor spaces
2culture bottle in, cultivate according to a conventional method, namely with containing the aminoacid of volumetric concentration 10% hyclone and culture medium (DMEM) culture fluid of glucose, put 37 DEG C, CO
2volumetric concentration is cultivate in the constant temperature and humidity incubator of 5%.When cell is in exponential phase, with mass concentration 0.25% trypsinization, collect Digestive system, centrifugal rear removal supernatant, makes cell suspension after washing 2 times with Hanks liquid, regulates cell concentration to be that every 5 μ l are containing 2.5 × 10
5the suspension of individual U87-MG cell is used for nude inoculation.
(2) modeling, grouping and administration:
Water is can't help in preoperative nude mice fasting, and after mass concentration 0.8% pentobarbital (0.2ml/10g) intraperitoneal injection of anesthesia, its head is fixed on stereo brain orienting instrument by ventricumbent position, after volumetric concentration 75% alcohol disinfecting head skin, prior to opening a vertical incision after cranium midline palpebral fissure, expose bregma, lmm before bregma mid point, sagittal suture to the right other 2.5mm place diameter of opening is that the animal cranial drill of 1mm carefully drills skull, then the microsyringe of 5 μ l tumor cell suspensions is had vertically to enter in alba district through hole with suction, depth of needle is that needle point is apart from skull surface 3.5mm, before injection, pin is return about 0.5mm a little, with 0.5 μ l/min injection speed, tumor cell suspension is slowly injected, slowly pin is pulled out again after stopping 2min before pin, bone hole is closed with bone wax, normal saline flushing visual area, sew up scalp, finally use iodine disinfection.
Conventional raising, observes its animation every day, comprises the situations such as spirit, diet, defecation, body weight and activity.After modeling after 14 days, choose the nude mice that Imaging-PAM in nuclear magnetic resonance scanning technology and living animal body all confirms successful tumor modeling, be divided into six groups at random, often organize 15, give the BU-SCP (0.05mg/ml, 0.1mg/ml and 0.2mg/ml) of low middle and high concentration respectively with vein according to administration volume/nude mice body weight (0.1ml/10g), BU-S (0.1mg/ml), positive drug temozolomide (TMZM, 2.5mg/ml) and solvent, every day 1 time, successive administration 10 days, often group stays 6, observation band tumor life cycle.All the other first use fluorescence imaging and nuclear magnetic resonance scanning in living animal body respectively at the 2d after drug withdrawal, detect signal intensity and the gross tumor volume of each group of nude mice in-vivo tumour, then sacrificed by decapitation, be separated tumor, get part tumor tissue 10% formaldehyde to fix, conventional embedding wax block, section carries out HE dyeing, light Microscopic observation tumor cell quantity, density and downright bad situation.
(3) experimental result is as follows:
1, the change of different dosing group nude mice body weight.
Result as shown in Figure 4, modeling is after 14 days, nude mouse weight average significantly increases (P<0.01), and often organize nude mouse weight average after administration and have decline in various degree, and high dose BU-SCP group and positive drug group body weight significantly decline (p<0.05 and p<0.01).This may be because every day intravenously administrable be a kind of external irritant concerning nude mice, in addition antineoplastic agent itself may have certain side effect, therefore administration starts nude mice body weight has and alleviates trend, but high dose BU-SCP group does not have positive drug group decline degree high, illustrate that the toxic and side effects of BU-SCP may be less than positive drug group.
2, the band tumor life cycle of different dosing group nude mice.
Result as shown in Figure 5, band tumor life cycle of administration group nude mice is all than contrast group leader, there is significant difference (P<0.01), and during same dose, BU-SCPmiddle group is with tumor life span extension (P<0.05) than BU-S group.
3, in living animal body, fluorescence imaging detects the signal intensity of each group of nude mice in-vivo tumour.
The fluorescence intensity that in living animal body, fluorescence imaging detects and the quantity of tumor cell have extraordinary linear relationship, can be reflected the quantity of cell by fluorescence intensity.As shown in Figure 6, successive administration is after 10 days, all remarkable than the negative control group minimizing (p<0.01) of the fluorescence intensity that each administration group detects, the fluorescence intensity ratio BU-S group of the BU-SCP group of same dose is low, and has statistical significance (P<0.05).Show that BU-SCP plays offect of ntiglioma in vivo, in the scope of data of experiment, become dose dependent, the offect of ntiglioma of BU-SCP is stronger than BU-S.
4, the gross tumor volume of different dosing group nude mice and tumour inhibiting rate
(1) comparison of gross tumor volume in different dosing group nude mice brain
Result as shown in Figure 7, the tumor nuclear-magnetism volume of administration group is all less, BU-SCPlow group and negative control group ratio have significant difference (p<0.05), other administration groups (BU-SCPmiddle group, BU-SCPhigh group, BU-S and TMZM) and negative control group have significant difference (P<0.01) than all, and BU-SCPmiddle group and BU-S group are than there being significant difference (P<0.05); BU-SCPhigh group and TMZM group are than the size no difference of science of statistics of tumor.
(2) each administration group tumour inhibiting rate compares
As shown in Figure 8, the tumour inhibiting rate of administration group and negative control group have significant difference (p<0.01) than tumour inhibiting rate to result; BU-SCPmiddle group and BU-S group have significant difference (P<0.05) than tumour inhibiting rate; BU-SCPhigh group and TMZM group are than tumour inhibiting rate no difference of science of statistics.
The effect that conclusion: BU-S and BU-SCP all has anti-cerebral glioma to grow, BU-SCP group in scope of experiment to the suppression of tumor in dose dependent.Isodose BU-SCP is stronger than BU-S offect of ntiglioma.
5, the histopathologic slide of transplanted tumor
Result as shown in Figure 9, HE dyeing and normal cerebral tissue's ratio, Glioma Model group cell dense arrangement, become bulk to assemble, nucleus is comparatively large and deeply, become infiltrative growth, cell atypia is obvious, nuclear division is visible mutually, and tumor tissue vascularization is irregular, and thin vessels is agglomerating to be present in around tumor tissue.BU-SCPlow group glioma cell pencil dense arrangement, visible tumor giant cell and obviously pathologic mitosis phase, new vessels enriches, visible little focal necrosis.BU-SCPmiddle and BU-S group is still shown in a small amount of pathologic mitosis picture, occurs the coagulation necrosis of stove shape, and downright bad composition comparatively BU-SCPlow group increases, and angiogenesis is obvious.BU-SCPhigh and TMZM group is downright bad in sheet, cellularity disintegrate, only remaining a small amount of cell and nuclear particulate.Illustrate that administration group all can kill tumor cell, and BU-SCP administration group increases with drug level, neoplasm necrosis composition increases, and volume reduces gradually.
6, the immunohistochemical staining of transplanted tumor
To the capable immunohistochemical staining of the tumor tissue of modeling after 14 days, detect the amynologic label Protein S-100 of brain tumor diagnosis and the specific expressed Protein G FAP of Astrocytic glioma, the expression of S-100 and GFAP lays respectively on endochylema and karyon, all in brown color, as shown in Figure 10 and Figure 11, the expression of results of S-100 with GFAP all becomes positive, in conjunction with bioluminescence image formation and nuclear magnetic resonance scanning result, shows nude mice modeling success.
7, BU-SCP induces the apoptosis of U87-MG cell
Respectively with 10,30, after 60ug/mlBU-SCP acts on U87-MG cell 24h, AnnexinV/PI analyzes each medication group apoptosis rate and is respectively (9.6% ± 1.1) %, (22.2% ± 1.7) %, (26.9% ± 2.4) %, each group of apoptosis rate is apparently higher than blank group (0.5% ± 0.02) the % same period, each administration group comparatively matched group has significant difference (P<0.01), illustrate that the apoptosis rate of each administration group raises with the increase of drug dose, BU-SCP is by the apoptosis killer cell of induction U87-MG cell.
Experimental example 8:
Inventor BU-NPP replaces BU-SCP to re-start the experimental procedure of experimental example 5-7, and result shows: the result of BU-NPP with BU-SCP is consistent.The drug effect of BU-NPP and BU-SCP can equivalence.
Anti-cerebral glioma medicine based on Venenum Bufonis extract of the present invention and preparation method thereof, it is respectively bufotalin submicron emulsion, bufotalin nano particle preparations.These two kinds of preparations all can be combined by the endotheliocyte in cerebral capillary, medicine permeable membrane is transmitted into brain, or gulp down transhipment effect by absorption mediation bag, improve the brain targeting of medicine, thus the distribution of bufotalin in brain can be made to increase further, the effect of anti-cerebral glioma is played by the apoptosis of inducing cell and excessive autophagy.
Although be described embodiment of the present invention above, the present invention is not limited to above-mentioned specific embodiments, and above-mentioned specific embodiments is only schematic, guiding, instead of restrictive.Those of ordinary skill in the art is under the enlightenment of this description, and when not departing from the scope that the claims in the present invention are protected, can also make a variety of dosage forms such as bufotalin liposome etc., these all belong to the row of the present invention's protection.
Claims (10)
1. the anti-cerebral glioma medicine based on Venenum Bufonis extract, for bufotalin submicron emulsion preparation, it is characterized in that, its 100mL standard dose component is: 50mg Venenum Bufonis extract, 10g medium chain triglyceride, 3g lecithin, 0.2g PLURONICS F87 (F-68), 2.5g glycerol, 0.05g enuatrol, all the other are water for injection.
2., according to claim 1 based on the anti-cerebral glioma medicine of Venenum Bufonis extract, it is characterized in that, in described Venenum Bufonis extract, the comprehensive content of Toadpoison Medicine, cinobufagin and bufogenin is greater than 95%.
3. a preparation method for bufotalin submicron emulsion preparation, is characterized in that, the preparation of its 100mL standard dose comprises the following steps:
(1) by 3g lecithin and 50mg Venenum Bufonis extract, add in 10g medium chain triglyceride respectively, heated and stirred is dissolved, and obtains the oil phase of the pastille clarified;
(2) add in water for injection by 0.2g PLURONICS F87 (F-68), 0.05g enuatrol and 2.5g glycerol, at 70 DEG C, heated and stirred makes dissolving obtain aqueous phase;
(3) under the stirring of high speed dispersor, oil phase is added in aqueous phase, continues to stir, obtained colostrum;
(4) colostrum is used hydrochloric acid solution adjust ph, be settled to 100mL with water for injection, cooling, is transferred to homogenizing in high pressure homogenizer, bottling, inflated with nitrogen, sealing, autoclaving, in psychrolusia cooling after taking-up, to obtain final product.
4. the preparation method of bufotalin submicron emulsion preparation according to claim 1, it is characterized in that, in step (1), the temperature of described heating is 78 DEG C, and the time is 30min.
5. the preparation method of bufotalin submicron emulsion preparation according to claim 1, it is characterized in that, in step (3), the rotating speed of described high speed dispersor is 12,000rpm, and mixing time is 3min.
6. the preparation method of bufotalin submicron emulsion preparation according to claim 1, it is characterized in that, in step (4), the concentration of described hydrochloric acid solution is 0.1molL
-1, pH value is between 5 ~ 6.5.
7. the preparation method of bufotalin submicron emulsion preparation according to claim 1, it is characterized in that, in step (4), the pressure of high pressure homogenizer is 800bar, homogenizing 8 times.
8. the preparation method of bufotalin submicron emulsion preparation according to claim 1, it is characterized in that, in step (4), autoclave temperature is 121 DEG C, and the time is 10min.
9., based on an anti-cerebral glioma medicine for Venenum Bufonis extract, be bufotalin nano particle preparations, it is characterized in that, its 100mL standard dose component is: 50mg Venenum Bufonis extract, 2.0 glyceryl monostearates, 0.8g medium chain length fatty acid triglyceride, 0.5g oleic acid, 2.0g lecithin
1.5g PLURONICS F87,0.3g NaTDC, all the other are water for injection; Wherein, in described Venenum Bufonis extract, the comprehensive content of Toadpoison Medicine, cinobufagin and bufogenin is greater than 95%.
10. a preparation method for bufotalin nano particle preparations, is characterized in that, comprises the following steps:
(1) take 50mg Venenum Bufonis extract, 2.0g glyceryl monostearate, 0.8g medium chain length fatty acid triglyceride, 0.5g oleic acid, heating in water bath lower magnetic force stirring and dissolving, obtain transparent homogeneous oil phase;
(2) by 2.0g lecithin
1.5g PLURONICS F87 (PluronicF68), 0.3g NaTDC is added in water for injection, and heat under magnetic agitation, dispersing and dissolving obtains aqueous phase;
(3) under magnetic stirring, be added dropwise to by aqueous phase while hot in identical temperature oil phase, ultrasonic disperse, under stirring condition, ice-water bath cools, and under aseptic condition, uses filter membrane Entkeimung, to obtain final product.
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| CN108464970A (en) * | 2018-04-25 | 2018-08-31 | 广州暨南生物医药研究开发基地有限公司 | A kind of anwulignan nano-emulsion and preparation method thereof of metabolism and the double suppressive of outer row |
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