CN107536826A - The method that self-assembly method prepares resveratrol dimer δ viniferin nanometer formulations - Google Patents
The method that self-assembly method prepares resveratrol dimer δ viniferin nanometer formulations Download PDFInfo
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Abstract
The invention discloses a kind of method that self-assembly method prepares resveratrol dimer δ viniferin nanometer formulations.By pluronic and resveratrol dimer mixed dissolution in organic solvent, mixed solution is at the uniform velocity instilled in the physiological saline of high-speed stirred, the nanoparticle dispersion liquid for carrying medicine is obtained using self-assembly method direct construction；Dialysis removes solvent and other soluble small molecules；Freeze drying protectant is added into nanoparticle dispersion liquid, obtains nanometer formulation solution；Again by freeze-drying by the moisture removal in nanometer formulation solution.This method can overcome the shortcomings of that resveratrol dimer δ viniferin have poorly water-soluble, improve its decentralization in water, significantly increase its stability in vivo, effectively improve bioavilability, play therapeutic effect.The preparation method is simple and reliable, has extraordinary application prospect.
Description
Technical field
The present invention relates to a kind of preparation technology of nanometer formulation, and in particular to a kind of self-assembly method prepares resveratrol dimerization
The method of body δ-viniferin nanometer formulations.
Background technology
Resveratrol dimer is the oligomer that resveratrol is formed by 4- hydroxystilbene peroxidase oxidizations, is mainly had
The resveratrol dehydrodimerization body of two kinds of structures of δ-viniferin and ε-viniferin.Wherein δ-viniferin are to downy mildew
The lethality of bacterium is 6 times of ε-viniferin, it is considered to be grape resists the important resistance marker of downy mildew.δ-viniferin
With a variety of pharmacological activities, including anti-inflammatory, anti-oxidant, antitumor, anti-diabetic and cardiovascular protective effect.Wei Yahui etc. is ground
Study carefully discovery, compared with resveratrol, δ-viniferin have stronger vasodilative effect, can significantly increase diameter of normal mouse auricula
Fine arteriovenous bore, improve microcirculation, positive therapeutic action can be played microcirculation disorder.However, δ-viniferin by
It is larger in molecular weight, body is reduced to its assimilation effect.In addition, solubility of the drug molecule in aqueous phase, is that medicine passes through
Intestines and stomach, into blood, reach the guarantee of intracorporeal organ and cell.However, solubility of the δ-viniferin in water is extremely low,
The bioavilability of the compound is caused to lower.Therefore, it is water-soluble that δ-viniferin are improved, so as to strengthen its bioavilability
It is that δ-viniferin exploitations must solve the problems, such as cardiovascular drugs.
Nano-medicament carrier can effectively improve solubility and internal stability of the hydrophobic drug in water, in extension body
Circulation time, targeted delivery is realized, be to overcome slightly solubility so as to reach the purpose for improving bioavilability, reducing toxic side effect
Medicine uses one of important method of obstacle.In addition nano-medicament carrier is also big with specific surface area, and surface reaction activity is high, easily
In modification many advantages, such as.Amphipathic nature material can be by being self-assembly of the nano-micelle of core shell structure, and kernel is hydrophobicity
Ultra microstructure, hydrophobic drug can be wrapped up, and shell is then the hydrophilic skeleton structure by some polysaccharide or high molecular polymer
Into because hydrophilic section has biocompatibility, to the three-dimensional stabilization of nano-particle.Such nano-medicament carrier can pass through
The method of self assembly prepares.
F68The block copolymer being made up of polyoxyethylene and polyethenoxy ether, be it is a kind of it is non-from
Subtype amphipathy macromolecule compound, there are many advantages such as low, the non-stimulated anaphylaxis of good biocompatibility, toxicity.Wherein
Five kinds of specifications of L44, F68, F87, F108, F127 have been ratified to carry out clinical practice, the preparation available for a variety of formulations by FDA.
Glen S.Kwon et al. have applied for preparing the patent (patent No. of F68 micella using membrane formation process：
US2012032115A1).But this method is not used to mass produce, reason is that film forming must be completed in container, causes
Film-forming amount is than relatively low, and different batches have the hidden danger in terms of quality control.At home, Sha Xianyi et al. using pluronic with
Insoluble drug methotrexate (MTX) carries out chemical crosslinking and obtains copolymer, then is prepared with pluronics material mixing and carry medicine glue
Beam (application number：201110287095.6), but this method material preparation process is complicated, and encapsulation process, which remains, needs film forming to go
Fall redisperse after organic solvent.Zhan Yong China et al. is prepared for resveratrol nanometer formulation, and applies obtaining a patent
(CN103142457A), but blank is still had in terms of resveratrol dimer δ-viniferin nanometer formulations.
Due to dissolubility extreme differences of the resveratrol dimer δ-viniferin in water, cause its biology profit in vivo
Expenditure is very low.Meanwhile resveratrol dimer δ-viniferin easily flocculate in water.Therefore, in pluronic encapsulation process
And after lyophilized formulations weight is molten, it is difficult to obtain stable small particle nano particle.
The content of the invention
It is an object of the invention to provide a kind of self-assembly method to prepare resveratrol dimer δ-viniferin nanometer formulations
Method.
To reach above-mentioned purpose, present invention employs following technical scheme：
This prepares the method for resveratrol dimer δ-viniferin nanometer formulations, comprises the following steps (following steps one
To four general progress at room temperature)：
Pluronic, resveratrol dimer δ-viniferin mixed dissolutions in organic solvent, are used fat by step 1
Dissolubility membrane filtration removes impurity, and mixed solution is made；Wherein, pluronic:Resveratrol dimer δ-viniferin:Have
Mass volume ratio (the mg of solvent:mg:ML it is) 20:10:1-2；
Above-mentioned resveratrol dimer δ-viniferin and pluronic mixed solution are scattered in physiology by step 2
In salt solution, obtain nanometer medicine and carry particle dispersion (nanoparticle dispersion liquid for carrying medicine)；Wherein, pluronic:Physiological saline
Mass volume ratio (mg:ML it is) 1:0.5-1；
Nanometer medicine is carried particle dispersion and loaded in molecular cut off >=3.0kDa bag filter by step 3, utilizes physiology
Dialysis against saline at least 12 hours, remove organic solvent and other soluble impurities；
Freeze drying protectant is scattered in the nanometer medicine by dialysis and carried in particle dispersion by step 4, is filtered using water solubility
Film removes other impurities, with physiological saline constant volume, obtains nanometer formulation solution；Wherein, freeze drying protectant:The mass ratio of pluronic
For 1-3:1；
Nanometer formulation solution is freeze-dried by step 5, obtains nanometer formulation dry powder.
Preferably, in the step 1), pluronic:Resveratrol dimer δ-viniferin:The quality of organic solvent
Volume ratio is 20:10:1；In the step 2), pluronic:The mass volume ratio of physiological saline is 1:0.5-0.6 is (optimal to be
1:0.5)；In the step 4), freeze drying protectant:The mass ratio of pluronic is 1-2:1 (optimal is 2:1), freeze drying protectant
Dosage is excessive, can cause that after nanometer formulation dry powder weight is molten stable nanoparticulate dispersion can not be formed, dosage is very few, influences to freeze
Dry effect, it is unformed powdered (shoulding normally be porous block) to cause lyophilized formulations, and is unfavorable for nanoparticle formulations
Weight is molten.
Preferably, the method for preparing resveratrol dimer δ-viniferin nanometer formulations, specifically includes following step
Suddenly：
Step 1 weighs 40-120 milligrams pluronic, 20-60 milligram resveratrol dimer δ-viniferin respectively
Mixed dissolution removes impurity in 2-15 milliliter organic solvents, and with the fat-soluble membrane filtration that aperture is 0.22 micron, is made
Mixed solution；
Described pluronic is selected from five specifications of L44, F68, F87, F108 or F127；
Described organic solvent is selected from can be with one kind in dimethyl sulfoxide (DMSO) miscible with water, dimethylformamide, ethanol
Or a variety of mixture；
Step 2 is by the mixed solution of resveratrol dimer δ-viniferin and pluronic with 1-2 ml/mins
Speed be slowly dropped into the physiological saline under 20-60 milliliter high-speed stirred states, mixing speed be 1000-1500 revs/min,
Obtain nanometer medicine and carry particle dispersion；
Nanometer medicine is carried particle dispersion and loaded in the bag filter that molecular cut off is 3.5kDa by step 3, utilizes physiology
Dialysis against saline 12-24 hours, during which change physiological saline (outer liquid) more than 3 times；
Step 4 weighs 80-240 milligram freeze drying protectants, the receiving by dialysis being slowly added under quick stirring
Rice medicine is carried in particle dispersion, 150-400 revs/min of mixing speed, treats that freeze drying protectant recycles aperture after being completely dissolved
Other impurities are removed for 0.22 micron of water-soluble filter membrane, are finally settled to 40-120mL with physiological saline, obtain nanometer formulation
Solution, it can so ensure that freeze-dried drug concentration can reach and contain 5 milligrams of resveratrol dimers in 10mL nanometer formulation solution
δ-viniferin；
Described freeze drying protectant is one or more kinds of mixed in mannitol, glucose, HES
Compound；
Step 5 dispenses nanometer formulation solution even, pre-freeze 24 hours at a temperature of≤- 70 DEG C, recycles freezing dry
Dry machine removes moisture removal, obtains final nanometer formulation dry powder.
The part nanometer formulation dry powder sterilizing pure water dissolving (weight is molten) that the present invention is obtained, utilizes Malvern ParticleSizer
Its particle diameter is determined, analyzes its performance.
Beneficial effects of the present invention are embodied in：
The present invention constructs one under water environment using pluronic parcel resveratrol dimer δ-viniferin
There can be nanoscale particle diameter with high degree of dispersion, medicine is active unaffected in itself, and granule stability is good, and encapsulation ratio is high to be received
Metrical scale preparation.It is complicated for most pluronic formulation ingredients, the problem of preparation technology is cumbersome, the present invention using it is several can be with
The organic solvent that water dissolves each other together dissolves medicine and packaging material, and is built directly in water by being self-assembly of nano-particle
Vertical process system is simple and practical, and each step quality control is accurate, can obtain higher encapsulation ratio and drugloading rate, prepared
Nanometer drug carrier system can stablize storage more than 1 year.The inventive method is easy, efficient, steady quality, can overcome resveratrol
The deficiency of dimer existing poorly water-soluble in itself, improves its decentralization in water, significantly increases its stability in vivo,
Bioavilability is effectively improved, is pushed further into its application level.Proportioning between adjunct ingredient and composition that the present invention utilizes
It is to ensure that nano particle is applied in vivo, produces the key of good result.
Further, by membrane filtration bacterium and the impurity particle of the other influences quality of the pharmaceutical preparations, to ensure follow-up use
Security and preparation stability in itself.
Further, the ratio embodied in technique is to grope optimization by many experiments to obtain, according to these ginsengs
Number, which prepare, can obtain stable nanometer formulation.
Further, used organic solvent toxicity is low.
Brief description of the drawings
Fig. 1 is the process chart of the present invention.
Fig. 2 is that pluronic prepared by embodiment 1 wraps up resveratrol dimer δ-viniferin nanometer formulation decentralization
Compare, wherein A is the nanometer formulation solution before freezing, and B is that resveratrol dimer δ-viniferin aqueous dispersions compare
Group, nanometer formulation solution of the C for dry powder weight after molten；Resveratrol dimer δ-viniferin concentration is identical in three kinds of samples.
Fig. 3 be pluronic prepared by embodiment 1 wrap up resveratrol dimer δ-viniferin nanometer formulations weight it is molten after
Granularmetric analysis result (B) after transmission electron microscope photo (A) and weight are molten.
Fig. 4 is that pluronic prepared by embodiment 1 wraps up resveratrol dimer δ-viniferin nanometer formulation solution grains
Footpath stability test result.
Fig. 5 is that pluronic prepared by embodiment 1 wraps up resveratrol dimer δ-viniferin nanometer formulations (14mg/
Kg, intraperitoneal injection, 1 times/day) induction (0.3 μm ol/ grams of Hcy, is subcutaneously injected, 2 times/day, induce 1 week) to homocysteine
Hyperhomocysteinemiainjury (HHcy) hypertensive rat blood pressure influence experimental result, wherein SBP is systolic pressure, and DBP is
Diastolic pressure.
Fig. 6 is that pluronic prepared by embodiment 1 wraps up resveratrol dimer δ-viniferin nanometer formulations (14mg/
Kg, intraperitoneal injection, 1 times/day) to the height of homocysteine (0.3 μm ol/ grams, be subcutaneously injected, 2 times/day, induces 1 week) induction
Plasma homocysteine (HHcy) regulation and control blood pressure target molecule (vascular smooth muscle A type endothelin receptors, i.e. ETA) expression experiment
As a result (β-actin are internal reference).
Embodiment
The present invention is described in detail with reference to the accompanying drawings and examples.
The present invention is wrapped using nonionic surfactant pluronic to resveratrol dimer δ-viniferin
Wrap up in, prepare nano level medicine and carry particle.Specifically utilize organic solvent such as dimethylformamide, diformazan that can be miscible with water
Base sulfoxide, ethanol etc. are by resveratrol dimer δ-viniferin and lapping pluronic co-dissolve, then will mix molten
Liquid is slowly dropped in the physiological saline of high-speed stirred, and directly obtaining nanometer medicine by the method for self assembly carries particle dispersion,
The step of middle film forming, redisperse etc. cause mass discrepancy or influence yield is saved, is removed divide using the method for dialysis afterwards
Organic solvent and soluble impurity in dispersion liquid, then add protective agent, finally by freeze-drying by the moisture in solution
Remove, so as to obtain end-product --- medicine (resveratrol dimer δ-viniferin) nanometer formulation dry powder (Fig. 1).
Embodiment 1
Step 1 weighs 40 milligrams of pluronic F127,20 milligrams of resveratrol dimer δ-viniferin mixing respectively
It is dissolved in 2 milliliters of dimethyl sulfoxide (DMSO)s, and impurity is removed with the fat-soluble membrane filtration that aperture is 0.22 micron, it is molten that mixing is made
Liquid.
Mixed solution is slowly dropped into the physiology under 20 milliliters of high-speed stirred states by step 2 with the speed of 1 ml/min
In salt solution, 1000 revs/min of mixing speed, obtain nanometer medicine and carry particle dispersion.
Nanometer medicine is carried particle dispersion and loads molecular cut off in the bag filter of 3500 dalton, to utilize by step 3
Normal saline dialysis 24 hours, during which change liquid 3 times, remove organic solvent and other soluble impurities in dispersion liquid.
Step 4 weighs 80 milligrams of mannitol, the load of the nanometer medicine by dialysis being slowly added under quick stirring
In grain dispersion liquid, mixing speed is 200 revs/min, waits the water-soluble filter for recycling aperture to be 0.22 micron after being completely dissolved
Film removes other impurities, final to be settled to 40 milliliters using physiological saline, obtains nanometer formulation solution.
Step 5 dispenses nanometer formulation solution even, pre-freeze 24 hours at a temperature of -70 DEG C, recycles freeze-drying
Machine (pressure is less than 0.1 pa) removes moisture removal, obtains nanometer formulation dry powder.
The nanometer formulation dry powder that the present embodiment is obtained is dissolved again with sterilizing pure water, determined using Malvern ParticleSizer
Resveratrol dimer δ-viniferin medicine-carried nano particles average grain diameter is 88 ± 3.6nm, and encapsulation ratio is 97 ± 2.1%.
Reference picture 2, Fig. 3 and Fig. 4 are visible, and resveratrol dimer δ-viniferin dispersivenesses in water are very poor, and liquid is muddy
It is turbid, have obvious sediment after standing, again dissolve after resveratrol dimer δ-viniferin nanometer formulations, than freeze it
Preceding nanometer formulation particle diameter slightly increase, but solution integrally it is limpid uniformly, stably；Illustrate that the present embodiment is wrapped up using pluronic
Points of the water-insoluble medicine resveratrol dimer δ-viniferin in water can significantly be increased by preparing nanometer medicine load particle
Divergence, particle diameter also comply with treatment needs.
Embodiment 2
Step 1 weighs 80 milligrams of pluronic F108,40 milligrams of resveratrol dimer δ-viniferin mixing respectively
It is dissolved in 4 milliliters of ethanol, and impurity is removed with the fat-soluble membrane filtration that aperture is 0.22 micron, mixed solution is made.
Mixed solution is slowly dropped into the physiology under 40 milliliters of high-speed stirred states by step 2 with the speed of 1 ml/min
In salt solution, 1500 revs/min of mixing speed, obtain nanometer medicine and carry particle dispersion.
Nanometer medicine is carried particle dispersion and loads molecular cut off in the bag filter of 3500 dalton, to utilize by step 3
Normal saline dialysis 24 hours, during which change liquid 3 times, remove solvent and other soluble impurities in dispersion liquid.
Step 4 weighs 160 milligrams of glucose, the load of the nanometer medicine by dialysis being slowly added under quick stirring
In grain dispersion liquid, mixing speed is 150 revs/min, waits the water-soluble filter for recycling aperture to be 0.22 micron after being completely dissolved
Film removes other impurities, final to be settled to 40 milliliters using physiological saline, obtains nanometer formulation solution.
Step 5 dispenses nanometer formulation solution even, pre-freeze 24 hours at a temperature of -70 DEG C, recycles freeze-drying
Machine (pressure is less than 0.1 pa) removes moisture removal, obtains nanometer formulation dry powder.
The nanometer formulation dry powder sterilizing pure water dissolving that the present embodiment is obtained, white lamb's-quarters is determined using Malvern ParticleSizer
The average grain diameter of reed alcohol dimer δ-viniferin medicine-carried nano particles is 89 ± 5.1nm, and encapsulation ratio is 98 ± 1.1%, solution
It is overall it is limpid uniformly, stably.
Embodiment 3
Step 1 weighs 120 milligrams of pluronic F87,60 milligrams of resveratrol dimer δ-viniferin mixing respectively
It is dissolved in 6 milliliters of ethanol, and impurity is removed with the fat-soluble membrane filtration that aperture is 0.22 micron, mixed solution is made.
Mixed solution is slowly dropped into the physiology under 60 milliliters of high-speed stirred states by step 2 with the speed of 2 ml/mins
In salt solution, 1500 revs/min of mixing speed, obtain nanometer medicine and carry particle dispersion.
Nanometer medicine is carried particle dispersion and loads molecular cut off in the bag filter of 3500 dalton, to utilize by step 3
Normal saline dialysis 24 hours, during which change liquid 3 times, remove solvent and other soluble impurities in dispersion liquid.
Step 4 weighs 240 milligrams of HESs, the nanometer medicine by dialysis being slowly added under quick stirring
Carry in particle dispersion, mixing speed is 400 revs/min, and it is 0.22 micron water-soluble that aperture is recycled after waiting to be completely dissolved
Property filter membrane remove other impurities, be finally settled to 40 milliliters using physiological saline, acquisition nanometer formulation solution.
Step 5 dispenses nanometer formulation solution even, pre-freeze 24 hours at a temperature of -70 DEG C, recycles freeze-drying
Machine (pressure is less than 0.1 pa) removes moisture removal, obtains nanometer formulation dry powder.
The nanometer formulation dry powder sterilizing pure water dissolving that the present embodiment is obtained, white lamb's-quarters is determined using Malvern ParticleSizer
The average grain diameter of reed alcohol dimer δ-viniferin medicine-carried nano particles is 86 ± 6.2nm, and encapsulation ratio is 96 ± 3.2%, solution
It is overall it is limpid uniformly, stably.
Embodiment 4
Step 1 weighs 80 milligrams of Pluronic F68s, 40 milligrams of resveratrol dimer δ-viniferin mixing respectively
It is dissolved in 4 milliliters of dimethylformamides, and impurity is removed with the fat-soluble membrane filtration that aperture is 0.22 micron, mixing is made
Solution.
Mixed solution is slowly dropped into the physiology under 40 milliliters of high-speed stirred states by step 2 with the speed of 2 ml/mins
In salt solution, 1000 revs/min of mixing speed, obtain nanometer medicine and carry particle dispersion.
Nanometer medicine is carried particle dispersion and loads molecular cut off in the bag filter of 3500 dalton, to utilize by step 3
Normal saline dialysis 24 hours, during which change liquid 3 times, remove solvent and other soluble impurities in dispersion liquid.
Step 4 weighs 160 milligrams of mannitol and glucose mixture (mass ratio=1:1), it is slowly added to quickly stir
The nanometer medicine by dialysis under state is carried in particle dispersion, and mixing speed is 300 revs/min, is waited after being completely dissolved again
Other impurities are removed using the water-soluble filter membrane that aperture is 0.22 micron, it is final to be settled to 40 milliliters using physiological saline, obtain
Nanometer formulation solution.
Step 5 dispenses nanometer formulation solution even, pre-freeze 24 hours at a temperature of -70 DEG C, recycles freeze-drying
Machine (pressure is less than 0.1 pa) removes moisture removal, obtains nanometer formulation dry powder.
The nanometer formulation dry powder sterilizing pure water dissolving that the present embodiment is obtained, white lamb's-quarters is determined using Malvern ParticleSizer
The average grain diameter of reed alcohol dimer δ-viniferin medicine-carried nano particles is 86 ± 3.3nm, and encapsulation ratio is 97 ± 1.5%, solution
It is overall it is limpid uniformly, stably.
Embodiment 5
Step 1 weighs 80 milligrams of pluronic L44,40 milligrams of resveratrol dimer δ-viniferin mixing respectively
It is dissolved in 4 milliliters of dimethyl sulfoxide (DMSO)s, and impurity is removed with the fat-soluble membrane filtration that aperture is 0.22 micron, it is molten that mixing is made
Liquid.
Mixed solution is slowly dropped into the physiology under 40 milliliters of high-speed stirred states by step 2 with the speed of 2 ml/mins
In salt solution, 1500 revs/min of mixing speed, obtain nanometer medicine and carry particle dispersion.
Nanometer medicine is carried particle dispersion and loads molecular cut off in the bag filter of 3500 dalton, to utilize by step 3
Normal saline dialysis 24 hours, during which change liquid 3 times, remove solvent and other soluble impurities in dispersion liquid.
Step 4 weighs 160 milligrams of mannitol and hydroxyethyl starch mixtures (mass ratio=1:1), it is slowly added to quick
The nanometer medicine by dialysis under stirring is carried in particle dispersion, and mixing speed is 200 revs/min, waits to be completely dissolved it
Recycle the water-soluble filter membrane that aperture is 0.22 micron to remove other impurities afterwards, be finally settled to 40 milliliters using physiological saline,
Obtain nanometer formulation solution.
Step 5 dispenses nanometer formulation solution even, pre-freeze 24 hours at a temperature of -70 DEG C, recycles freeze-drying
Machine (pressure is less than 0.1 pa) removes moisture removal, obtains nanometer formulation dry powder.
The nanometer formulation dry powder sterilizing pure water dissolving that the present embodiment is obtained, white lamb's-quarters is determined using Malvern ParticleSizer
The average grain diameter of reed alcohol dimer δ-viniferin medicine-carried nano particles is 88 ± 3.7nm, and encapsulation ratio is 95 ± 3.8%, solution
It is overall it is limpid uniformly, stably.
Embodiment 6
Step 1 weighs 120 milligrams of pluronic F127 respectively, 60 milligrams of resveratrol dimer δ-viniferin are mixed
Conjunction is dissolved in 6 milliliters of ethanol, and removes impurity with the fat-soluble membrane filtration that aperture is 0.22 micron, and mixed solution is made.
Mixed solution is slowly dropped into the physiology under 60 milliliters of high-speed stirred states by step 2 with the speed of 2 ml/mins
In salt solution, 1500 revs/min of mixing speed, obtain nanometer medicine and carry particle dispersion.
Nanometer medicine is carried particle dispersion and loads molecular cut off in the bag filter of 3500 dalton, to utilize by step 3
Normal saline dialysis 24 hours, during which change liquid 3 times, remove solvent and other soluble impurities in dispersion liquid.
Step 4 weighs 240 milligrams of glucose and hydroxyethyl starch mixtures (mass ratio=1:1), it is slowly added to quick
The nanometer medicine by dialysis under stirring is carried in particle dispersion, and mixing speed is 400 revs/min, waits to be completely dissolved it
Recycle the water-soluble filter membrane that aperture is 0.22 micron to remove other impurities afterwards, be finally settled to 40 milliliters using physiological saline,
Obtain nanometer formulation solution.
Step 5 dispenses nanometer formulation solution even, pre-freeze 24 hours at a temperature of -70 DEG C, recycles freeze-drying
Machine (pressure is less than 0.1 pa) removes moisture removal, obtains nanometer formulation dry powder.
The nanometer formulation dry powder sterilizing pure water dissolving that the present embodiment is obtained, white lamb's-quarters is determined using Malvern ParticleSizer
The average grain diameter of reed alcohol dimer δ-viniferin medicine-carried nano particles is 89 ± 2.4nm, and encapsulation ratio is 95 ± 3.5%, solution
It is overall it is limpid uniformly, stably.
Embodiment 7
Step 1 weighs 40 milligrams of pluronic F127,20 milligrams of resveratrol dimer δ-viniferin mixing respectively
It is dissolved in 2 milliliters of dimethyl sulfoxide (DMSO)s and alcohol mixeding liquid (volume ratio=1:1) in, and it is 0.22 micron of fat-soluble filter with aperture
Membrane filtration removes impurity, and mixed solution is made.
Mixed solution is slowly dropped into the physiology under 20 milliliters of high-speed stirred states by step 2 with the speed of 2 ml/mins
In salt solution, 1000 revs/min of mixing speed, obtain nanometer medicine and carry particle dispersion.
Nanometer medicine is carried particle dispersion and loads molecular cut off in the bag filter of 3500 dalton, to utilize by step 3
Normal saline dialysis 24 hours, during which change liquid 3 times, remove solvent and other soluble impurities in dispersion liquid.
Step 4 weighs 80 milligrams of mannitol and glucose mixture (mass ratio=2:1), it is slowly added to quickly stir shape
The nanometer medicine by dialysis under state is carried in particle dispersion, and mixing speed is 200 revs/min, is waited sharp again after being completely dissolved
Other impurities are removed with the water-soluble filter membrane that aperture is 0.22 micron, finally 40 milliliters is settled to using physiological saline, is received
Metric system agent solution.
Step 5 dispenses nanometer formulation solution even, pre-freeze 24 hours at a temperature of -70 DEG C, recycles freeze-drying
Machine (pressure is less than 0.1 pa) removes moisture removal, obtains nanometer formulation dry powder.
The nanometer formulation dry powder sterilizing pure water dissolving that the present embodiment is obtained, white lamb's-quarters is determined using Malvern ParticleSizer
The average grain diameter of reed alcohol dimer δ-viniferin medicine-carried nano particles is 89 ± 5.1nm, and encapsulation ratio is 97 ± 2.5%, solution
It is overall it is limpid uniformly, stably.
The present invention wraps up prepared pluronic the particle diameter point of resveratrol dimer δ-viniferin nanometer formulations
Cloth and stability are measured, and pass through its morphological feature of transmission electron microscope observation, the results showed that：The nanometer formulation has
Very high stability, strong to medicine parcel ability, morphological feature is homogeneous, and monodispersity is good.And to resveratrol dimer
Anti- blood pressure ability is detected inside δ-viniferin nanometer formulations, and effect experiment result finds that the nanometer formulation can have
Effect reduces blood pressure (Fig. 5).Show that it significantly reduces the egg of A type endothelin receptors by Western Blot experimental results
White expression (Fig. 6), has good therapeutic effect.By contrast, resveratrol dimer δ-viniferin direct administrations can not
Reach above-mentioned therapeutic effect.
The present invention has found that conventional medical or food nonionic surface active agent such as tween and this class are through overtesting
Row can not form stable resveratrol dimer δ-viniferin nanometer formulations, and the holding time is shorter, it is impossible to prepare lyophilized
Agent.Although the nanometer formulation that ionic surfactant is formed is relatively stable, it is secondary to there is larger poison when applying in vivo
Effect, while bioavilability is relatively low.And F68 packaging material used in the present invention can effectively overcome resveratrol dimer
Deficiency present in the preparation of δ-viniferin nanometer formulations.Wherein crucial reagent matches, preparation technology is by a large amount of real
Test and grope what is obtained, required nanometer formulation can not be obtained by directly imitating the technological parameter of other pertinent literatures.In addition, auxiliary material
It is another pith for ensureing that the quality of the pharmaceutical preparations is stable, because there is the participation of adjunct ingredient, in enough drugloading rates and encapsulation ratio
On the premise of, determine relevant parameter and the technological process of auxiliary material addition finally by many experiments, overcome it in preparation process
Present in stability deficiency and repeatability it is poor the shortcomings that, ensure that the quality control of preparation.
Claims (10)
1. the method that self-assembly method prepares resveratrol dimer δ-viniferin nanometer formulations, it is characterised in that：This prepares white
The method of veratryl alcohol dimer δ-viniferin nanometer formulations, comprises the following steps：
1) with fat-soluble filter membrane mistake after pluronic and resveratrol dimer δ-viniferin being mixed in organic solvent
Filter, obtains mixed solution；Wherein, pluronic:Resveratrol dimer δ-viniferin mass ratio is 2:1, it is general per 20mg
Lang Nike is dissolved in 1-2mL organic solvents；
2) mixed solution is scattered in physiological saline, obtains nanometer medicine and carry particle dispersion；Wherein, per 1mg pluronics point
Dissipate in 0.5-1mL physiological saline；
3) nanometer medicine is carried after particle dispersion loads in molecular cut off >=3.0kDa bag filter and utilizes normal saline dialysis
At least 12 hours；
4) freeze drying protectant is scattered in after nanometer medicine by dialysis is carried in particle dispersion with water-soluble membrane filtration, incited somebody to action
Filter gained filtrate obtains nanometer formulation solution with physiological saline constant volume；Wherein, freeze drying protectant:The mass ratio of pluronic is 1-
3:1。
2. the method that self-assembly method according to claim 1 prepares resveratrol dimer δ-viniferin nanometer formulations,
It is characterized in that：It is described prepare resveratrol dimer δ-viniferin nanometer formulations method it is further comprising the steps of：It will receive
Metric system agent solution is freeze-dried, and obtains nanometer formulation dry powder.
3. the method that self-assembly method according to claim 1 prepares resveratrol dimer δ-viniferin nanometer formulations,
It is characterized in that：In the step 1), 1mL organic solvents are dissolved in per 20mg pluronics；It is general per 1mg in the step 2)
Lang Nike is scattered in 0.5-0.6mL physiological saline；In the step 4), freeze drying protectant:The mass ratio of pluronic is 1-2:
1。
4. the method that self-assembly method according to claim 1 prepares resveratrol dimer δ-viniferin nanometer formulations,
It is characterized in that：The method for preparing resveratrol dimer δ-viniferin nanometer formulations specifically includes following steps：
1) by 40-120 milligrams pluronic, 20-60 milligram resveratrol dimer δ-viniferin mixed dissolutions in 2-15 millis
Rise in organic solvent, then with the fat-soluble membrane filtration that aperture is 0.22 micron, obtain mixed solution；
2) mixed solution is added in the physiological saline under 20-60 milliliter stirrings with the speed of 1-2 ml/mins, stirring
Speed is 1000-1500 revs/min, obtains nanometer medicine and carries particle dispersion；
3) nanometer medicine is carried into particle dispersion to load in the bag filter that molecular cut off is 3.5kDa, utilizes normal saline dialysis
12-24 hours；
4) 80-240 milligrams freeze drying protectant is added into being carried by the nanometer medicine dialysed in particle dispersion under stirring, stirred
150-400 revs/min of speed is mixed, treats to use the water-soluble membrane filtration that aperture is 0.22 micron after freeze drying protectant dissolving, so
40-120 milliliters are settled to physiological saline afterwards, obtain nanometer formulation solution；
5) nanometer formulation solution is freeze-dried, obtains nanometer formulation dry powder.
5. the method that self-assembly method according to claim 1 prepares resveratrol dimer δ-viniferin nanometer formulations,
It is characterized in that：The pluronic is selected from L44, F68, F87, F108 or F127.
6. the method that self-assembly method according to claim 1 prepares resveratrol dimer δ-viniferin nanometer formulations,
It is characterized in that：One or more kinds of mixing of the organic solvent in dimethyl sulfoxide (DMSO), dimethylformamide, ethanol
Thing.
7. the method that self-assembly method according to claim 1 prepares resveratrol dimer δ-viniferin nanometer formulations,
It is characterized in that：The freeze drying protectant is one or more kinds of mixed in glucose, mannitol, HES
Compound.
8. the method that self-assembly method according to claim 2 prepares resveratrol dimer δ-viniferin nanometer formulations,
It is characterized in that：The condition of the freeze-drying is：Pre-freeze 24 hours at a temperature of≤- 70 DEG C, freeze drier is recycled to go
Moisture removal.
9. the method that self-assembly method as claimed in claim 1 prepares resveratrol dimer δ-viniferin nanometer formulations exists
Prepare the application in antihypertensive drugs.
10. the method that self-assembly method as claimed in claim 1 prepares resveratrol dimer δ-viniferin nanometer formulations exists
Prepare for reduce A type endothelin receptor protein expressions medicine in application.
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