CN1391889A - Tanshinone nano preparation and its preparing method - Google Patents
Tanshinone nano preparation and its preparing method Download PDFInfo
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Abstract
The co-fused nano powder preparation of tanshinone and polyglycol 4000-6000 is fast soluble in physiological saline to form colloid liquid capable of being stored stably. Its preparation process includes heating at 70-90 deg.C to fuse polyglycol 4000-6000, mixing fused polyglycol 4000-6000 and tanshinone to form the co-fused material, cooling, grinding and 200-300 mesh sieved.
Description
Technical field
The present invention relates to tanshinone nano preparation and preparation method thereof, especially nanometer formulation of tanshinone, Tanshinone I and preparation method thereof.
Background technology
The three dimensional structure of TANSHINONES and the energy function of molecule have determined it to be the high-melting-point material, and are insoluble in water.For example: the fusing point of tanshinone is 485.7K, and the fusing point of Tanshinone I is 495.4K, and the fusing point of cryptotanshinone is that 464.6K is (referring to " thermodynamic study of TANSHINONES homologue " " China Medicine University's journal " 1988; 19 (2): 90-92).The hydrophilic/hydrophobic index of tanshinone is 79/401=0.1970, and the hydrophilic/hydrophobic index of Tanshinone I is 80/377=0.2122.Their dissolubility in water had both depended on the hydrophilic/hydrophobic index, the three dimensional structure that also depends on them, for being one, the Tanshinone I of aromatic ring approaches complete planar molecule as the A ring, more be insoluble in water, in a single day these fat-soluble compounds enter in the body fluid, will produce hydrophobic interaction (referring to J.Fisher ﹠amp under the driving of hydrone; J.R.P. Arnold (Li Yanmei translates) " chemistry in biology " pp.86-89 Science Press 2000 Beijing).With fat-soluble solvent such as ethanol etc. from red rooted salvia Salviamiltiorrhiza easily extraction separation to the crystalloid TANSHINONES (referring to Luo Houwei Wu BaojingYong Zhongen et al. Pigments from Salvia miltiorrhiza " Phytochemistry " 1,985 24 (4) 815-817), this crystal habit and its distribute in plant or the physical form that comes across in the Chinese medicine decoction has very big difference.The physical form and its pharmacologically active that have experiment to show that TANSHINONES exists have substantial connection.
Over nearly 10 years,, cause new knowledge to the TANSHINONES pharmacological action because increasing " novel targets " found in the development of Celluar and Molecular Biology.Under 0.5 μ g/ml, 5 routine acute promyelocytic leukemia (APL) patients are had induction of differentiation as tanshinone, to human hepatoma cell strain SMMC-7721 also have reverse and short differentiation (referring to: tanshinones such as the refined orchid of beam bright Yuan of brave sheep descendants are induced promyelocytic leukemia cell differentiation and Study on Molecular Mechanism " Chinese Journal of Hematology " 200021 (1) 23-26 thereof; The refined blue yellow tough quick king of Yuan repaiies the reverse effect " tumor " 1997,17 (5) of TANSHINONES such as outstanding person to some phenotype of human liver cancer cell: 268-270).The scholar of Korea S also finds tanshinone leukaemia to the people under 1 μ g/ml dosage, and HL60 and K562 all can cause apoptosis by activation Caspase enzyme.
But, because the hydrophilic index of TANSHINONES is so low, makes water soluble preparation and have difficulties, above-mentioned numerous bioactive discoveries are and adopt elder generation that TANSHINONES is dissolved in dimethyl sulfoxine (DMSO), the then method of dilute with water by a certain percentage.Dimethyl sulfoxine can cause several animal crystalline lens muddinesses, and its whole body is used the approval that does not obtain administration of health department.
Also have the tanshinone sulfonation is made sodium tanshinone IIA sulfate salt, and be used for 108 routine patients with coronary heart disease, discovery can alleviate angina pectoris and improve the ischemic electrocardiogram (referring to Shanghai Cooperative group for thestudy of Tanshinone IIA " J.Traditional Chinese Medicine " 1984,4:20).Because its half-life in blood is short, snap shot and slow phase were respectively 26 and 108 minutes, therefore must with 160mg/250ml/ day this heavy dose carry out intravenous drip (referring to:
35Sulfur-sodium tanshinone IIA sulfate is drained and metabolic research in the intravital distribution of rat, " Chinese patent medicine research " 1979,2:8-12).In addition, sulfonate sodium is an ionic compound, and is difficult by the biofilm structure system based on lipid and protein component.Bacteriostatic activity to tulase experimental results show that, tanshinone and tanshinone sulfonate, its MIC is respectively 0.31 μ g/ml and 〉=5 μ g/ml, fungistatic effect differs greatly (referring to the thick luxuriant high big Zheng Jia profit of recording of: sieve, the structure activity relationship of tanshinone and allied compound bacteriostasis " China Medicine University's journal " 1988,19 (4): 258-262).
Disclose a kind of TANSHINONES micropowder preparation among the CN00119579.4, TANSHINONES is carried out microwave radiation earlier, grind jointly with hydrophilicity condiment again, make TANSHINONES turn to amorphous state, to promote its dissolution from crystalline state by microwave radiation.
Summary of the invention
Technical scheme of the present invention is as follows:
The technical problem to be solved in the present invention is that development a kind ofly can be treated the especially leukemic TANSHINONES preparation of human diseases, the used adjuvant of said preparation must meet the standard of human drugs, bioavailability improves, can keep TANSHINONES can be smoothly intrinsic propesties by cell membrane, can be dispersed in the aqueous medium again, be convenient to clinical injection or intravenous drip.
The technical problem to be solved in the present invention comprises that also the preparation method of described TANSHINONES preparation can keep and promote not only that the pharmacologically active of TANSHINONES preparation in the prior art manifests, and should be able to replace the TANSHINONES preparation of preparing the back dilute with water with DMSO, and its activity is significantly improved.The preparation method of said preparation should be in industrial application simultaneously.
For solving the problems of the technologies described above, technical scheme of the present invention is:
A kind of tanshinone nano preparation is characterized in that: by weight, the fused matter powder by TANSHINONES 5.0～10% and Macrogol 4000 or polyethylene glycol 6000 90～95% are formed is the precursor of nanometer solution.
According to aforementioned tanshinone nano preparation, it is characterized in that: TANSHINONES is 5.0～10% tanshinones or 5.0～10% Tanshinone Is; Fused matter powder with polyethylene glycol 6000 90～95% compositions.
Described tanshinone nano preparation is characterized in that: the fused matter powder that TANSHINONES 5.0～10% and Macrogol 4000 or polyethylene glycol 6000 90～95% are formed, can be dissolved in normal saline, and form nanometer colloid.
Described tanshinone nano preparation is characterized in that: fused matter nanometer powder, be dissolved in normal saline, and place after 1 day, form the nanometer colloid of colloidal solid less than 100 nanometers.
The tanshinone nano preparation preparation method is characterized in that: by weight, get TANSHINONES 5.0～10% and Macrogol 4000 or 6,000 90～95%; Earlier Macrogol 4000 or 6000 is heated to 70～90 ℃ of thawings;
In Macrogol 4000 that melts or polyethylene glycol 6000, add TANSHINONES, stir, form TANSHINONES-Polyethylene Glycol fused matter; Grind the cooling back, crosses 200～300 mesh sieves.
Preparation method according to described tanshinone nano preparation is characterized in that: by weight, get tanshinone or Tanshinone I 5.0～10%, polyethylene glycol 6000 is 90～95%; After earlier polyethylene glycol 6000 being heated to 70～90 ℃ of thawings; Add tanshinone or Tanshinone I and stir, form tanshinone-Polyethylene Glycol fused matter or Tanshinone I-Polyethylene Glycol fused matter; After the cooling, use ball mill grinding,, ground 1～4 hour, cross 300 mesh sieves, be dissolved in normal saline, after the placement, form the nanometer colloid of colloidal solid less than 100 nanometers with 350～550 rev/mins rotating speed.
Preparation method according to described tanshinone nano preparation is characterized in that: by weight, get tanshinone or Tanshinone I 5.0～10%, polyethylene glycol 6000 is 90～95%; After earlier polyethylene glycol 6000 being heated to 80 ℃ of thawings; Add tanshinone or Tanshinone I and stir, form tanshinone-Polyethylene Glycol fused matter or Tanshinone I-Polyethylene Glycol fused matter; After the cooling, use ball mill grinding,, ground 2.5～3.5 hours, cross 300 mesh sieves, be dissolved in normal saline, after the placement, form the nanometer colloid of colloidal solid less than 100 nanometers with 400～500 rev/mins rotating speed.
Preparation method according to described tanshinone nano preparation is characterized in that: by weight, get tanshinone or Tanshinone I 5.0～10%, polyethylene glycol 6000 is 90～95%; After earlier polyethylene glycol 6000 being heated to 80 ℃ of thawings; Add tanshinone or Tanshinone I and stir, form tanshinone-Polyethylene Glycol fused matter or Tanshinone I-Polyethylene Glycol fused matter; After the cooling, use ball mill grinding,, ground 3 hours, behind 300 mesh sieves, be dissolved in normal saline excessively, place after 1 day, form the nanometer colloid of colloidal solid less than 100 nanometers with 450 rev/mins rotating speed.
In the preparation method of aforementioned tanshinone nano preparation, use the electron microscope observation nano-sized colloidal solution, it is characterized in that: sample dyes without the negative staining dyestuff.
Described TANSHINONES, tanshinone, Tanshinone I are Radix Salviae Miltiorrhizae extract.
The definition of at present relevant " nanoparticle " is: the microgranule that can see with ultramicroscope is called nanoparticle.
The present invention screens taking polyethylene glycol 4000～6000 as the solid hydrophilic dispersant, the corresponding single fused matter that it can form with TANSHINONES, tanshinone or Tanshinone I, and not only fusing point is low, and possess hydrophilic property.Experiment shows that its ratio shared in fused matter is big more, and the dispersion of tanshinone composition is just big more, and this helps reducing intermolecular nonbonding active force, comprises that Van der Waals interacts and electrostatic force.
After making the low melting point fused matter, cooling curing is ground into it about 0.5cm again
3Agglomerate, use QM-1SP2/2L type planetary ball mill (Nanjing Univ. Instrument Factory's manufacturing) to grind 2～4 hours then with 350-550 rev/min rotating speed, finish grinding after, cross 200～300 mesh sieves, micropowder after sieving is through observation by light microscope, and its big I reaches micron/submicron order.TANSHINONES, tanshinone or Tanshinone I and Macrogol 4000 or 6000 fused matters that form, after grinding, granularity is more small, and specific surface area increases.When TANSHINONES: the fused matter micropowder that Macrogol 4000～6000 form when being 5.0～10%:90～95%, but the dispersion moment in water finish, and, form less than the colloidal attitude granule below 100 nanometers by hydrophobic interaction.This colloidal solution bin stability is good.
Utilization differential scanning heat instrument (model: NETZSCH DSC 204), measured under the different condition, formed the fusing point of single TANSHINONES-Polyethylene Glycol fused matter.For example: parameter in contrast, the tanshinone fusing point is 218.5 ℃, referring to: Figure 11; When dispersant accounts for 95%, cross the 5% tanshinone-polyethylene glycol 6000 fused matter of 300 mesh sieves, fusing point drops to 58.9 ℃, referring to: Figure 12; Cross the 5% tanshinone-polyethylene glycol 6000 fused matter of 200 mesh sieves, fusing point drops to 59.1 ℃, referring to: Figure 13; Cross the 10% tanshinone-polyethylene glycol 6000 fused matter of 200 mesh sieves, fusing point drops to 59.7 ℃, referring to: Figure 14.And in to tanshinone-above-mentioned test of polyethylene glycol 6000 fused matter, temperature programming one all finds no the thermogram that fusing point is 218.5 ℃ a single tanshinone until 250 ℃.
In the above-mentioned experiment, fusing point descends and to reach Δ=159.5 ℃, show with Polyethylene Glycol-6000 as solid dispersion, can reduce the non-bond energy between the tanshinone molecule, can Yin Wendu not too high and cause the variation or the structural deterioration of chemical constituent, make the tanshinone and the fused matter of Polyethylene Glycol-6000 formation have strong hydrophilic simultaneously again.And above-mentioned experiment also shows: melting altogether in the body, do not having the crystallization of TANSHINONES, but forming the fused matter of tanshinone and Polyethylene Glycol-6000.
The tanshinone crystalline solid also can make its micronization by simple grinding, but because its very high hydrophobicity can not make itself and water form colloid solution.What we studied once that the tanshinone crystallization occurred in the normal saline dilution of methanol solution and different proportion is transformed into the dynamic change of nanoparticle by " micro-crystallization ".Get the tanshinone of crystalline state, by methanol: TANSHINONES=2ml: 2mg is mixed with storing solution and deposits, and uses Ringer ' s normal saline to press storing solution and Ringer ' s normal saline 1: 1 then; 1: 5; Be mixed with sample with 1: 50 three kinds of different proportion, respectively at the 1st day, got above-mentioned sample in the 5th day and the 10th day and dye method for making sample routinely (referring to: turbulent waves chief editor " biomedical ultrastructure and Electron Microscopy " Science Press, 1980 p177 Beijing), be prepared into transmission electron microscope sample, with the method difference be that sample is not done dying operation, reason is that the contrast of formed contrast of negative staining dyestuff and TANSHINONES colloidal solid is very approaching, the image of TANSHINONES colloidal solid can be covered.
Under the Ringer ' of different proportion s normal saline condition, form the process of colloidal solid with the transmission electron microscope observation tanshinone, experimental result is as follows: 1, the tanshinone crystalline powder is dispersed in to remain with crystalline state in a period of time behind the methanol and exists, its two-dimensional is about 1.65 μ m (wide) * 4.4 μ m (length), be in it and compare crystalline size under solid state and be reduced to the micron number magnitude, this process approximately sustainable about 10 days.2, the ratio of Ringer ' s normal saline has direct influence to the formation of crystalline rate of dispersion of TANSHINONES and micelle, and the normal saline ratio increases as in 1: 50, can accelerate the formation of crystalline rate of dispersion of TANSHINONES and micelle.3, being bar-shaped TANSHINONES crystalline particle and when its width is in 50-60nm, will no longer being dispersed into littler crystalline particle in disperse medium, but begin to lose the distinctive form of crystallization, gradually disperse to be formed diameter less than the micelle of 5nm and be accumulated into agglomerate, microgranule in agglomerate and then gather and form the micelle that diameter is about 20nm, be accumulated into diameter again and be about the 60nm micelle, this process is approximately by a definite date about 10 days, but be subjected to the scale effect of Ringer ' s normal saline, the water ratio increases the formation that can accelerate crystalline rate of dispersion of TANSHINONES and micelle.4, the feature that diameter is about the 60nm micelle is to be connected into chain each other in turn and to twine rich configuration, and wherein Ge Ti feature is an edge clear size rule, is oval spherical, diameter is about 60nm, and oval spheric major axis is the direction that is connected with each other along micelle.More than test explanation TANSHINONES crystallite passes through hydrophobic interaction in normal saline, can form the nano-sized colloidal solution of diameter, but need to place overlong time 10 days less than the colloidal solid of 100 nanometers, be not easy to clinical use, and methanol is toxic does not allow to be used for human body.The more important thing is that when the tanshinone crystalline powder was dispersed in the methanol with microcrystalline state, experiment in vitro was observed and do not demonstrated due pharmacologically active; Have only when it forms nano colloidal particles, just present pharmacologically active.
Observe the image of colloidal particles under transmission electron microscope that tanshinone-Polyethylene Glycol-6000 fused matter forms with transmission electron microscope (TEM).Get the micropowder preparation of tanshinone-Polyethylene Glycol-6000 eutectic through grinding, by 50 μ g/ml, 20 μ g/ml, the ratio of 2 μ g/ml, be mixed with solution with normal saline, left standstill 1 day, through hydrophobic interaction, above-mentioned solution is pressed preceding method, be prepared into transmission electron microscope sample, observe with transmission electron microscope, the result shows: preparation method of the present invention not only can reach the nano colloidal particles solution of micelle less than 100 nanometers, and greatly shortened time of colloid solution forming process, have more practicality.
The invention solves a kind of water solublity problem of unsolved TANSHINONES preparation of a specified duration, develop and a kind ofly can treat the especially leukemic TANSHINONES preparation of human diseases, the used adjuvant Polyethylene Glycol of preparation TANSHINONES-Polyethylene Glycol fusions meets the standard of human drugs, the bioavailability of said preparation improves, both having kept TANSHINONES can be dispersed in the aqueous medium again smoothly by the intrinsic propesties of cell membrane.After TANSHINONES-Polyethylene Glycol nanometer powder adds normal saline, can keep at least stablizing more than 400 hours, can be advantageously used in clinical injection or intravenous drip.
The technical problem that the present invention solves comprises that also the preparation method of described TANSHINONES preparation can keep and promote not only that the pharmacologically active of TANSHINONES preparation in the prior art manifests, and can replace the TANSHINONES system property of preparing the back dilute with water with DMSO fully, and its activity is significantly improved.The preparation method of said preparation can be in industrial application simultaneously.
Description of drawings
Fig. 1: tanshinone-Polyethylene Glycol-6000 2mg fused matter microgranule is dissolved in the 20ml normal saline, is prepared into the photo (enlargement ratio: 40000X) can see this eutectic granularity greatly about 0.20 * 0.05 μ m-5 * 0.5 mu m range that transmission electron microscope sample is observed immediately after the formation stock solution.
Fig. 2: when above-mentioned stock solution and normal saline are 1: 1 (V/V, down together), prepare the transmission electron microscope photo after 1 day, can see the tanshinone micelle and or form (enlargement ratio: 80000X)
Fig. 3: stock solution and normal saline are 1: 1 o'clock, prepare the transmission electron microscope photo after 1 day, can see tanshinone Polyethylene Glycol fused matter granularity greatly about 0.1 * 0.05-0.35 * 0.15 μ m, (enlargement ratio: 40000X).
Fig. 4: stock solution and normal saline are 1: 5 o'clock, prepare the transmission electron microscope photo after 1 day, and tanshinone Polyethylene Glycol micelle forms, and colloid diameter distributes and is about 30-100nm, and is interconnection between them, (enlargement ratio: 80000X)
Fig. 5: stock solution and normal saline are 1: 5, prepare the transmission electron microscope photo after 1 day, and tanshinone Polyethylene Glycol micelle forms, and colloid diameter distributes and to be about 25-50nm, they interconnection and twine rich, (enlargement ratio: 80000X)
Fig. 6: stock solution and normal saline are 1: 5 o'clock, prepare the transmission electron microscope photo after 1 day, and tanshinone Polyethylene Glycol micelle degree is greatly about 0.1 * 0.05-0.35 * 0.15 μ m, (enlargement ratio: 40000X)
Fig. 7: stock solution and normal saline are 1: 50, prepare the transmission electron microscope photo after 1 day, have many diameters gel particle distribution less than 5nm, and also having diameter is that 20nm is gel particle distribution, and minority is greater than 80nm micelle, (enlargement ratio: 80000X)
Fig. 8: stock solution and normal saline are 1: 50, prepare the transmission electron microscope photo after 1 day, have diameter gel particle distribution and be gathered into diameter and be about 60nm micelle (dark granule among the figure), (enlargement ratio: 80000X) less than 5nm
Fig. 9: stock solution and normal saline are 1: 50, prepare the transmission electron microscope photo after 1 day, and it is interconnection and winding, (enlargement ratio: 80000X) arranged that diameter is about the 60nm micelle
Figure 10: stock solution and normal saline are 1: 50, prepare the transmission electron microscope photo after 1 day, and diameter is about the 80nm micelle and forms (enlargement ratio: 80000X)
Figure 11: differential scanning heat instrument is measured the tanshinone melting-point diagram, and drawing shows that its fusing point is 218.5 ℃
Figure 12: differential scanning heat instrument was measured the 5% tanshinone-polyethylene glycol 6000 fused matter melting-point diagram of 300 mesh sieves, and drawing shows that its fusing point is 58.9 ℃,
Figure 13: differential scanning heat instrument was measured the 5% tanshinone-polyethylene glycol 6000 fused matter melting-point diagram of 200 mesh sieves, and drawing shows that its fusing point is 59.1 ℃.
Figure 14: differential scanning heat instrument was measured 200 mesh sieves, 10% tanshinone-polyethylene glycol 6000 fused matter melting-point diagram, and drawing shows that its fusing point is 59.7 ℃
Figure 15: high-efficient liquid phase technique is measured tanshinone-Polyethylene Glycol micropowder water liquid concentration change figure
Figure 16: high-efficient liquid phase technique is measured tanshinone-Polyethylene Glycol micropowder water liquid 400 hour concentration variation diagrams
The specific embodiment
The sample purity of tanshinone, Tanshinone I is 90 ± 5%, and its extraction separation method can be according to document Luo Houwei Wu Baojing Yong Zhongen et al.；Pigments?from?Salvia?miltiorrhizaPhytochemistry?1985?24(4)815-817。
Polyethylene Glycol-6000, about M.W.6000, Japanese import packing, Shanghai chemical reagent factory, lot number: 80-08-22.
Embodiment 1:
Get tanshinone 5 grams, Polyethylene Glycol-6,000 95 grams
At first make Polyethylene Glycol-6000 be heated to 80 ℃ and make its whole thawings, add tanshinone again and be stirred to crystalline solid and disappear and form till the uniform fused matter.After the cooling, use ball mill grinding,, ground 2.5 hours, cross 300 mesh sieves, be dissolved in normal saline, place after 1 day, form the nano-sized colloidal solution of colloidal solid less than 100 nanometers with 450 rev/mins rotating speed.
Embodiment 2:
Get Tanshinone I 5 grams, Polyethylene Glycol-4,000 95 grams
At first make Polyethylene Glycol-4000 be heated to 70 ℃ and make its whole thawings, add Tanshinone I again and be stirred to crystalline solid and disappear and form till the uniform fused matter.After the cooling, use ball mill grinding,, ground 3 hours, cross 300 mesh sieves, be dissolved in normal saline, place after 1 day, form the nano-sized colloidal solution of colloidal solid less than 100 nanometers with 350 rev/mins rotating speed.
Embodiment 3:
Get tanshinone 10 grams (purity is 90%), Polyethylene Glycol-4,000 185 grams
At first make Polyethylene Glycol-6000 be heated to 70 ℃ and make its whole thawings, add tanshinone again and be stirred to crystalline solid and disappear and form till the uniform fused matter.After the cooling, use ball mill grinding,, ground 1 hour, cross 200 mesh sieves, be dissolved in normal saline, place after 1 day, form the nano-sized colloidal solution of colloidal solid less than 100 nanometers with 550 rev/mins rotating speed.
Embodiment 4:
Get Tanshinone I 20 grams, Polyethylene Glycol-6,000 185 grams.
At first make Polyethylene Glycol-6000 be heated to 90 ℃ and make its whole thawings, add Tanshinone I again and be stirred to crystalline solid and disappear and form till the uniform fused matter.After the cooling, use ball mill grinding,, ground 2 hours, cross 300 mesh sieves, be dissolved in normal saline, after the placement, form the nano-sized colloidal solution of colloidal solid less than 100 nanometers with 400 rev/mins rotating speed.
Embodiment 5: except that the fusing point of the fused matter that measures tanshinone and PEG, do not see the fusing point of tanshinone in the DSC method, illustrate that tanshinone is disperseed fully by PEG institute.
The stability of colloid solution---the assay of tanshinone in the colloidal solution:
In order to investigate by TANSHINONES colloidal particles and aqueous solution by a certain percentage, under certain condition by the steady statue of formed colloid solution behind the hydrophobic interaction, adopt the high performance liquid chromatogram method to the tanshinone content in the colloid solution, made mensuration.
Take by weighing the tanshinone-Polyethylene Glycol micropowder 4.1mg of preparation among the embodiment 1, be dissolved in the 2.0ml water, wherein tanshinone content is 5%, and each sample size is 10 microlitres, according to concentration and peak area linear dependence equation, Y=1E+06+91318, Y wherein: peak area, X: concentration, correlation coefficient: R2=0.999, the concentration of trying to achieve is max=387microM, min=301microM.Experiment is carried out at twice, has measured in different intervals 13 times for the first time, and the concentration of having observed in 71 hours changes, and has measured in different intervals 12 times for the second time, has observed the concentration change in 400 hours, referring to Figure 15,16.The concentration of each point among Figure 16 is represented at peak among Figure 15.Concentration change in 400 hours illustrates that colloidal state is stable in 10% as seen from the figure.The actual conditions of measuring is as follows:
Beckman System Gold Liquid chromatograph is equipped with 125 Solvent modulepump and 166 UV-VIS detectors, and analytical column is 250 * 4.6mm Beckman Ultrasphere C
18(5 μ m).Mobile phase is by solvent orange 2 A (0.075% trifluoroacetic acid) and solvent B (acetonitrile) gradient elution 0-5min, 0%B; 5-25min, 0-70%B; 40-41min, the 70%B. flow keeps 1.0ml min
-1The mensuration wavelength is 280nm.
Embodiment 6:
Transmission electron microscope (TEM) is observed the image of colloidal particles under transmission electron microscope that tanshinone-Polyethylene Glycol-6000 fused matter forms.
Get the micropowder preparation of tanshinone-Polyethylene Glycol-6000 eutectic through grinding of preparation among the embodiment 1, by 50 μ g/ml, 20 μ g/ml, the ratio of 2 μ g/m is mixed with solution with normal saline, leaves standstill 1 day.Above-mentioned solution is pressed preceding method, be prepared into transmission electron microscope sample, observe with transmission electron microscope.
Experimental results show that: the fineness ratio of tanshinone-Polyethylene Glycol-6000 eutectic through grinding obviously reduces without the volume that grinds; The normal saline of different proportion is big or small influential to the micelle speed that forms and the micelle that forms; The sample of above-mentioned different proportion can both form micelle in the time after preparation, and particle diameter can reach 60nm.
Fig. 1 is prepared into the photo (enlargement ratio: 40000X) can see this eutectic granularity greatly about 0.20 * 0.05 μ m-5 * 0.5 mu m range that transmission electron microscope sample is observed immediately after being dissolved in the formation stock solution of 20ml normal saline with tanshinone-Polyethylene Glycol-6000 2mg eutectic microgranule.
Fig. 2 is a stock solution: normal saline=1: 1 o'clock, at the transmission electron microscope photo of preparation after 1 day, can see the tanshinone micelle or forms, differ in size, the about 100nm of wherein big colloid diameter, the about 60-20nm of less diameter does not wait, and is interconnection and winding, (enlargement ratio: 80000X) arranged between them
Fig. 3 is a stock solution: normal saline=1: 1 o'clock, at the transmission electron microscope photo of preparation after 1 day, can see tanshinone Polyethylene Glycol fused matter granularity greatly about 0.1 * 0.05-0.35 * 0.15 μ m, (enlargement ratio: 40000X).
Fig. 4 is a stock solution: normal saline=1: 5 o'clock, and at the transmission electron microscope photo of preparation after 1 day, tanshinone Polyethylene Glycol micelle forms, and colloid diameter distributes and is about 30-100nm, and is interconnection between them, (enlargement ratio: 80000X)
Fig. 5 is a stock solution: the transmission electron microscope photo of normal saline=1: 5 preparation after 1 day, tanshinone Polyethylene Glycol micelle forms, colloid diameter distributes and is about 25-50nm, they interconnection and twine rich, (enlargement ratio: 80000X)
Fig. 6 is a stock solution: normal saline=1: 5 o'clock, at the transmission electron microscope photo of preparation after 1 day, tanshinone Polyethylene Glycol micelle degree is greatly about 0.1 * 0.05-0.35 * 0.15 μ m, (enlargement ratio: 40000X)
Fig. 7 is a stock solution: the normal saline=transmission electron microscope photo of preparation in 1: 50 after 1 day, and there are many diameters gel particle distribution less than 5nm, also having diameter is that 20nm is gel particle distribution, and minority is greater than 80nm micelle, (enlargement ratio: 80000X)
Fig. 8 is a stock solution: the transmission electron microscope photo of normal saline=1: 50 preparation after 1 day has diameter gel particle distribution and be gathered into diameter and be about 60nm micelle (dark granule among the figure), (enlargement ratio: 80000X) less than 5nm
Fig. 9 is a stock solution: it is interconnection and winding, (enlargement ratio: 80000X) arranged that the transmission electron microscope photo of normal saline=1: 50 preparation after 1 day, diameter are about the 60nm micelle
Figure 10 is a stock solution: the normal saline=transmission electron microscope photo of preparation in 1: 50 after 1 day, diameter is about the 80nm micelle and forms (enlargement ratio: 80000X)
Embodiment 7:
Effect experiment
Entrust the Huaxi Hospital Attached to Sichuan Univ institute of oncology to carry out effect experiment.
Get 5% and 10% the tanshinone-PEG6000 fused matter micropowder preparation for preparing in the foregoing description, be dissolved in normal saline, dosage is for being converted to IIA 0.5 μ g/ml; Reference substance is the tanshinone 0.5 μ g/ml that is dissolved in DMSO.Research experimental results show that the external evoked differentiation of HL-60 cell line: 5% compares with reference substance with tanshinone-PEG6000 fused matter micropowder preparation of 10%, and its activity has remarkable improvement.
Claims (9)
1, a kind of tanshinone nano preparation is characterized in that: by weight, the fused matter powder by TANSHINONES 5.0～10% and Macrogol 4000 or polyethylene glycol 6000 90～95% are formed is the precursor of nanometer solution.
2, according to the described tanshinone nano preparation of claim 1, it is characterized in that: TANSHINONES is 5.0～10% tanshinones or 5.0～10% Tanshinone Is; Fused matter powder with polyethylene glycol 6000 90～95% compositions.
3, according to claim 1 or 2 described tanshinone nano preparations, it is characterized in that: the fused matter powder that TANSHINONES 5.0～10% and Macrogol 4000 or polyethylene glycol 6000 90～95% are formed, can be dissolved in normal saline, form nanometer colloid.
4, according to the described tanshinone nano preparation of claim 3, it is characterized in that: fused matter nanometer powder, be dissolved in normal saline, place after 1 day, form the nanometer colloid of colloidal solid less than 100 nanometers.
5, a kind of preparation method of tanshinone nano preparation is characterized in that:
By weight, get TANSHINONES 5.0～10% and Macrogol 4000 or polyethylene glycol 6000 90～95%;
Earlier Macrogol 4000 or polyethylene glycol 6000 are heated to 70～90 ℃ of thawings;
In Macrogol 4000 that melts or polyethylene glycol 6000, add TANSHINONES, stir, form TANSHINONES-Polyethylene Glycol fused matter;
Grind the cooling back, crosses 200～300 mesh sieves.
6, according to the preparation method of the described tanshinone nano preparation of claim 5, it is characterized in that: by weight, get tanshinone or Tanshinone I 5.0～10%, polyethylene glycol 6000 is 90～95%; After earlier polyethylene glycol 6000 being heated to 70～90 ℃ of thawings; Add tanshinone or Tanshinone I and stir, form tanshinone-Polyethylene Glycol fused matter or Tanshinone I-Polyethylene Glycol fused matter; After the cooling, use ball mill grinding,, ground 1～4 hour, cross 300 mesh sieves, be dissolved in normal saline, after the placement, form the nanometer colloid of colloidal solid less than 100 nanometers with 350～550 rev/mins rotating speed.
7, according to the preparation method of the described tanshinone nano preparation of claim 6, it is characterized in that: by weight, get tanshinone or Tanshinone I 5.0～10%, polyethylene glycol 6000 is 90～95%; After earlier polyethylene glycol 6000 being heated to 80 ℃ of thawings; Add tanshinone or Tanshinone I and stir, form tanshinone-Polyethylene Glycol fused matter or Tanshinone I-Polyethylene Glycol fused matter; After the cooling, use ball mill grinding,, ground 2.5～3.5 hours, cross 300 mesh sieves, be dissolved in normal saline, after the placement, form the nanometer colloid of colloidal solid less than 100 nanometers with 400～500 rev/mins rotating speed.
8, according to the preparation method of the described tanshinone nano preparation of claim 7, it is characterized in that: by weight, get tanshinone or Tanshinone I 5.0～10%, polyethylene glycol 6000 is 90～95%; After earlier polyethylene glycol 6000 being heated to 80 ℃ of thawings; Add tanshinone or Tanshinone I and stir, form tanshinone-Polyethylene Glycol fused matter or Tanshinone I-Polyethylene Glycol fused matter; After the cooling, use ball mill grinding,, ground 3 hours, behind 300 mesh sieves, be dissolved in normal saline excessively, place after 1 day, form the nanometer colloid of colloidal solid less than 100 nanometers with 450 rev/mins rotating speed.
9, according to the preparation method of the described tanshinone nano preparation of one of claim 6～8, use the electron microscope observation nano-sized colloidal solution, it is characterized in that: sample dyes without the negative staining dyestuff.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100457085C (en) * | 2005-05-26 | 2009-02-04 | 湖南大学 | Polyglycol coated nano plant extract prepn and its prepn process |
| CN117487154A (en) * | 2023-10-31 | 2024-02-02 | 暨南大学 | Tanshinone derivative and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100457085C (en) * | 2005-05-26 | 2009-02-04 | 湖南大学 | Polyglycol coated nano plant extract prepn and its prepn process |
| CN117487154A (en) * | 2023-10-31 | 2024-02-02 | 暨南大学 | Tanshinone derivative and preparation method and application thereof |
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