CN108567741A - Puerarin Liposomal formulation and preparation method thereof for treating atherosclerosis - Google Patents
Puerarin Liposomal formulation and preparation method thereof for treating atherosclerosis Download PDFInfo
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- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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Abstract
The present invention relates to Liposomal formulation fields, in particular to a kind of Puerarin Liposomal formulation and preparation method thereof for treating atherosclerosis.A kind of Puerarin Liposomal formulation for treating atherosclerosis, it is characterised in that：The preparation contains the component of following mass ratio：1 part of Puerarin, carrier phosphatidase 3~9 part, 1~6 part of surfactant, 3~10 parts of excipient, 3~10 parts of disintegrant.Puerarin Liposomal formulation pharmacological action of the present invention for treating atherosclerosis understands that reliably preparation process is relatively simple, and oral administration biaavailability is high and toxic side effect is small, is suitble to take for a long time, has good market promotion prospect.
Description
Technical field
The present invention relates to Liposomal formulation fields, in particular to a kind of Puerarin lipid for treating atherosclerosis
Body preparation and preparation method thereof.
Background technology
Atherosclerosis refer to have artery wall thickening, be hardened and elasticity reduce, and with endarterium formed atheromatous plaque
The main reason for lesion being characterized is coronary heart disease, cerebral infarction, peripheral vascular disease.It is in recent years most for the pathogenesis of the disease
Scholar supports " endothelial injuries reaction theory ", it is believed that the final all damaged arteries inner membrance of the various Major Risk Factors of the disease, and congee
The formation of sample hardening lesion is the result for inflammation-fibroproliferative reaction that artery makes inner film injury.
It can be dysfunction or anatomical injury that endarterium is impaired.In the case of Long-term High-fat mass formed by blood stasis, the fat egg that increases
Mainly the low-density lipoprotein (ox LDL) of oxidative modification and cholesterol cause functional to endarterium in white, make interior
Chrotoplast and leucocyte (monocyte and lymphocyte) surface characteristic change, and adhesion factor expression increases.Monocyte
The quantity being attached on endothelial cell increases, and becomes macrophage under inner membrance from being moved between endothelial cell, passes through street cleaner
Receptor swallows oxLDL, is changed into the earliest atherosclerotic lesion fatty streaks of foam wanshing.Macrophage can aoxidize LDL,
Form peroxide and super oxidative ionic, moreover it is possible at least six kinds of cell factors are synthesized and secrete, in the effect of these cell factors
Under, promote fatty streaks to develop into fiber fat lesion, developing deeply is fibrous plaque.
In the case where haemodynamics changes, as blood pressure increases, turbulent flow caused by blood vessel local stenosis and cuts
Stress variation etc. makes the continuity between endarterium endothelial cell interrupt, endothelial cell retraction, to subintimal group of exposure
It knits.Blood platelet in the blood of platelet activating factor (PAF) activation at this time, is allowed to stick, be gathered on inner membrance, form attached wall blood
Bolt.Blood platelet can disengage many cell factors.These factors enter arterial wall, also thin to inspiring smooth muscle in atherosclerotic lesion
Born of the same parents' hyperplasia plays an important role.
Atherosclerosis is prevalent in crowd, and different degrees of atherosclerosis can last for several years and even count
10 years and be not accompanied by any clinical symptoms.Atherosclerosis often involves aorta, coronary artery, cerebral artery and the arteria renalis, disease
Can occur that atherosclerotic plaque rupture, thrombosis, vessel lumen be narrow or occlusion in journey, to make the blood of related organ
Obstacle occurs for supply.In any case, it can be suddenly with the not similar shape such as myocardial infarction, cerebral apoplexy/TIA, local organization ischemic
Formula is broken out or even threat to life.Disease caused by atherosclerosis is the most common cause of death of developing country.
Atherosclerosis is clinically more common in 40 years old or more, the elderly, is in progress after 49 years old rapid, there is data sheet
It is bright, in 50 to 60 years old crowds, there is 77% people to have different degrees of artery sclerosis, Atherosclerosis in 60 years old or more crowd
Change incidence and is up to 79.9%.
The drug of common antiatherosclerosis is broadly divided into following a few classes currently on the market：Blood lipid-lowering medicine, anti-blood
Platelet drug, vasodilator drug, thrombus dissolving and anticoagulation medicine.Wherein, the blood lipid-lowering medicine of Statins is that current treatment is dynamic
The most frequently used drug of pulse atherosclerosis has the function of inhibiting human body synthetic cholesterol, reduces triglyceride concentration in blood, in short term
It inside takes safer, takes this medicine for a long time and then easy to produce side effect.So the patient for taking this medicine for a long time should inspect periodically
The projects such as its blood alanine aminotransferase and creatine kinase.
Puerarin is also known as puerarin, is the isoflavonoid derivatives with coronary dilatation effect detached from Chinese medicament kudzu-vine root,
With blood vessel is expanded, improve blood circulation, increase coronary flow, adjust blood microcirculation, reduces cardiovascular and cerebrovascular disease danger
Clinical effect can be used for the treatment of atherosclerosis.But Puerarin is slightly soluble in water, and oral administration biaavailability is extremely low, faces at present
Injection system administration is mostly used on bed, it is serious also to particular patient there may be the potential toxic side effect such as allergy, haemolysis
It can lead to death.
Invention content
Present invention aim to solve the deficiency of above-mentioned background technology, provide a kind of for treating atherosclerosis
Puerarin Liposomal formulation and preparation method thereof.
The technical scheme is that：A kind of Puerarin Liposomal formulation for treating atherosclerosis, feature
It is：The preparation contains the component of following mass ratio：1 part of Puerarin, carrier phosphatidase 3~9 part, surfactant 1~6
Part, 3~10 parts of excipient, 3~10 parts of disintegrant.
The further carrier phosphatide is soybean lecithin, hydrogenated soya phosphatide, lecithin, hydrolecithin, brain phosphorus
One kind in fat.
The further surfactant is polyethylene glycol -15- hydroxy stearic acid esters, Tween 80, PLURONICS F87
In one kind.
The further excipient is 30 POVIDONE K 30 BP/USP 25, PVP K30,30 POVIDONE K 30 BP/USP 60,30 POVIDONE K 30 BP/USP 90, poly- second two
One kind in alcohol 2000, Macrogol 4000, Macrogol 6000, PEG 8000.
The further disintegrant is crosslinked polyvinylpyrrolidone.
A kind of preparation method for treating the Puerarin Liposomal formulation of atherosclerosis, it is characterised in that：Including
Following steps：
1), example takes a Puerarin, 3~9 parts of carrier phosphatide, 1~6 part of surfactant, 3~10 parts of figurations in mass ratio
Agent and 3~10 portions of disintegrants, which are added to stirring in absolute ethyl alcohol, makes it fully dissolve and be uniformly mixed；
2), mixture is dried under reduced pressure to viscous liquid in stirring condition, disintegrant is added and is mixed with mixture
Uniformly；
3), continue to be dried under reduced pressure to absolute ethyl alcohol and completely remove totally, the solid matter of acquisition is crushed, is sieved i.e.
It can obtain the Liposomal formulation.
Advantages of the present invention has：1, Puerarin is slightly soluble in water, and oral administration biaavailability is very low (being less than 3%), and nanometer is made
After liposome, oral administration biaavailability (being more than 60%) is substantially increased；
2, Puerarin at present clinically is administered for injection system, and patient may occur in which the toxic side effects such as allergy, haemolysis, seriously
The advantages of person can lead to death, this invention is to improve therapeutic effect, reduces toxic side effect；
3, it recent studies have shown that, the generation of atherosclerosis is mainly due to the generation of lipid peroxide, Puerarin
The vascular endothelial cell being damaged by lipid peroxide can be protected, the therapy target of atherosclerosis is moved forward, disease is conducive to
The treatment and prevention of disease, to achieve the effect that reduce hypertension, hyperlipidemia occurs；
The preparation method technique of the present invention is relatively simple, and is nearly free from the three wastes, uses absolute ethyl alcohol as organic
Solvent, toxicity is extremely low and recyclable recycling, helps to control production cost.
Puerarin Liposomal formulation pharmacological action of the present invention for treating atherosclerosis understands reliably, prepares
Technique is relatively simple, and oral administration biaavailability is high and toxic side effect is small, is suitble to take for a long time, before having good marketing
Scape.
Specific implementation mode
The present invention is further explained in the light of specific embodiments.Illustrated embodiment is not as the limit to the present invention
It is fixed.If not specified, drug ingedient dosage is mass fraction.
Examples 1 to 4：Puerarin, soybean lecithin, polyethylene glycol -15- hydroxy stearates are taken according to the mass fraction in following table
Acid esters, PVP K30 and crosslinked polyvinylpyrrolidone,
1, Puerarin, soybean lecithin, polyethylene glycol -15- hydroxy stearic acid esters and PVP K30 are added sequentially to anhydrous
In ethyl alcohol, stirring makes it fully dissolve and be uniformly mixed；
2, it is dried under reduced pressure under stirring condition in viscous liquid, crosslinked polyvinylpyrrolidone is added and is uniformly mixed；
3, continue to be dried under reduced pressure to absolute ethyl alcohol to eliminate, crush, be sieved to obtain the final product.
Embodiment 5~8：Puerarin, lecithin, polyethylene glycol -15- hydroxy stearic acids are taken according to the mass fraction in following table
Ester, 30 POVIDONE K 30 BP/USP 60 and crosslinked polyvinylpyrrolidone,
1, Puerarin, lecithin, polyethylene glycol -15- hydroxy stearic acid esters and 30 POVIDONE K 30 BP/USP 60 are added sequentially to anhydrous second
In alcohol, stirring makes it fully dissolve and be uniformly mixed；
2, it is dried under reduced pressure under stirring condition in viscous liquid, crosslinked polyvinylpyrrolidone is added and is uniformly mixed；
3, continue to be dried under reduced pressure to absolute ethyl alcohol to eliminate, crush, be sieved to obtain the final product.
Embodiment 9~12：Puerarin, soybean lecithin, Tween 80, Macrogol 4000 are taken according to the mass fraction in following table
And crosslinked polyvinylpyrrolidone,
1, Puerarin, soybean lecithin, Tween 80 and Macrogol 4000 are added sequentially in absolute ethyl alcohol, stirring makes it
It fully dissolves and is uniformly mixed；
2, it is dried under reduced pressure under stirring condition in viscous liquid, crosslinked polyvinylpyrrolidone is added and is uniformly mixed；
3, continue to be dried under reduced pressure to absolute ethyl alcohol to eliminate, crush, be sieved to obtain the final product.
Embodiment 13：1 part of Puerarin, 3 parts of hydrogenated soya phosphatide, 1 part of PLURONICS F87, poly- dimension are taken according to mass fraction
3 parts of 1 part of ketone K25 and crosslinked polyvinylpyrrolidone,
1, Puerarin, hydrogenated soya phosphatide, PLURONICS F87 and 30 POVIDONE K 30 BP/USP 25 are added sequentially in absolute ethyl alcohol, are stirred
Mixing makes it fully dissolve and be uniformly mixed；
2, it is dried under reduced pressure under stirring condition in viscous liquid, crosslinked polyvinylpyrrolidone is added and is uniformly mixed；
3, continue to be dried under reduced pressure to absolute ethyl alcohol to eliminate, crush, be sieved to obtain the final product.
Embodiment 14：1 part of Puerarin, 4 parts of hydrolecithin, 2 parts of PLURONICS F87, povidone are taken according to mass fraction
4 parts of 4 parts of K90 and crosslinked polyvinylpyrrolidone,
1, Puerarin, hydrolecithin, PLURONICS F87 and 30 POVIDONE K 30 BP/USP 90 are added sequentially in absolute ethyl alcohol, are stirred
It is set fully to dissolve and be uniformly mixed；
2, it is dried under reduced pressure under stirring condition in viscous liquid, crosslinked polyvinylpyrrolidone is added and is uniformly mixed；
3, continue to be dried under reduced pressure to absolute ethyl alcohol to eliminate, crush, be sieved to obtain the final product.
Embodiment 15：1 part of Puerarin, 5 parts of cephalin, 3 parts of PLURONICS F87, polyethylene glycol are taken according to mass fraction
2000 5 parts and 5 parts of crosslinked polyvinylpyrrolidone,
1, Puerarin, cephalin, PLURONICS F87 and polyethylene glycol 2000 are added sequentially in absolute ethyl alcohol, are stirred
It is set fully to dissolve and be uniformly mixed；
2, it is dried under reduced pressure under stirring condition in viscous liquid, crosslinked polyvinylpyrrolidone is added and is uniformly mixed；
3, continue to be dried under reduced pressure to absolute ethyl alcohol to eliminate, crush, be sieved to obtain the final product.
Embodiment 16：1 part of Puerarin, 6 parts of hydrogenated soya phosphatide, 4 parts of PLURONICS F87, poly- second are taken according to mass fraction
6 parts of 6,000 6 parts of glycol and crosslinked polyvinylpyrrolidone,
1, Puerarin, hydrogenated soya phosphatide, PLURONICS F87 and Macrogol 6000 are added sequentially to absolute ethyl alcohol
In, stirring makes it fully dissolve and be uniformly mixed；
2, it is dried under reduced pressure under stirring condition in viscous liquid, crosslinked polyvinylpyrrolidone is added and is uniformly mixed；
3, continue to be dried under reduced pressure to absolute ethyl alcohol to eliminate, crush, be sieved to obtain the final product.
Embodiment 17：1 part of Puerarin, 7 parts of hydrolecithin, 5 parts of Tween 80, polyethylene glycol are taken according to mass fraction
8000 7 parts and 7 parts of crosslinked polyvinylpyrrolidone,
1, Puerarin, hydrolecithin, Tween 80 and PEG 8000 are added sequentially in absolute ethyl alcohol, stirring makes
It fully dissolves and is uniformly mixed；
2, it is dried under reduced pressure under stirring condition in viscous liquid, crosslinked polyvinylpyrrolidone is added and is uniformly mixed；
3, continue to be dried under reduced pressure to absolute ethyl alcohol to eliminate, crush, be sieved to obtain the final product.
Embodiment 18：According to mass fraction take 1 part of Puerarin, 8 parts of cephalin, 6 parts of Tween 80,25 8 parts of 30 POVIDONE K 30 BP/USP and
8 parts of crosslinked polyvinylpyrrolidone,
1, Puerarin, cephalin, Tween 80 and 30 POVIDONE K 30 BP/USP 25 are added sequentially in absolute ethyl alcohol, stirring makes it fully
It dissolves and is uniformly mixed；
2, it is dried under reduced pressure under stirring condition in viscous liquid, crosslinked polyvinylpyrrolidone is added and is uniformly mixed；
3, continue to be dried under reduced pressure to absolute ethyl alcohol to eliminate, crush, be sieved to obtain the final product.
Embodiment 19：1 part of Puerarin, 9 parts of hydrolecithin, 6 parts of PLURONICS F87, povidone are taken according to mass fraction
9 parts of 9 parts of K90 and crosslinked polyvinylpyrrolidone,
1, Puerarin, hydrolecithin, PLURONICS F87 and 30 POVIDONE K 30 BP/USP 90 are added sequentially in absolute ethyl alcohol, are stirred
It is set fully to dissolve and be uniformly mixed；
2, it is dried under reduced pressure under stirring condition in viscous liquid, crosslinked polyvinylpyrrolidone is added and is uniformly mixed；
3, continue to be dried under reduced pressure to absolute ethyl alcohol to eliminate, crush, be sieved to obtain the final product.
Embodiment 20：1 part of Puerarin, 9 parts of cephalin, 6 parts of PLURONICS F87, polyethylene glycol are taken according to mass fraction
2000 10 parts and 10 parts of crosslinked polyvinylpyrrolidone,
1, Puerarin, cephalin, PLURONICS F87 and polyethylene glycol 2000 are added sequentially in absolute ethyl alcohol, are stirred
It is set fully to dissolve and be uniformly mixed；
2, it is dried under reduced pressure under stirring condition in viscous liquid, crosslinked polyvinylpyrrolidone is added and is uniformly mixed；
3, continue to be dried under reduced pressure to absolute ethyl alcohol to eliminate, crush, be sieved to obtain the final product.
The quality examination of Puerarin nano liposomes
1, average grain diameter measures
One bag of gained Puerarin nano liposome preparations (containing Puerarin 100mg) in above-described embodiment is taken, water about 100ml is added
Make dissolving, selects the size distribution of laser scattering method this product.This product average grain diameter should be less than 500nm.
The average grain diameter result of three batches of samples is as follows：
2, the measurement of encapsulation rate
One bag of gained Puerarin nano liposome preparations (containing Puerarin 100mg) in above-described embodiment is taken, water 100ml is added to shake
It is even, it takes about 3.5ml to set in super filter tube, is centrifuged 30 minutes with 12000 revs/min, filtrate is measured according to the method under assay item
Middle drug content；It separately takes in the stoste 1ml to 10ml volumetric flasks not centrifuged, is diluted to scale with mobile phase, shakes up, filter, together
Method measures.With main peak area computational envelope rate, encapsulation rate should be greater than 80%.Encapsulation rate=[(stoste main peak area × 10- filtrates
Main peak area)/stoste main peak area × 10] × 100%.
The encapsulation rate result of three batches of samples is as follows：
3, assay
Assay index using the content of Puerarin as this product selects pueraria lobata in high effective liquid chromatography for measuring this product
The content of element.
Chromatographic condition：Chromatographic column of the C18 columns (4.6 × 250mm, 5 μm) as assay is selected, with methanol：Water (30:
70, v/v) it is used as mobile phase.Detection wavelength 250nm, 30 DEG C, flow velocity 1ml/min of column temperature, 20 μ l of sample size.
Content assaying method：
The preparation of External standards solutions：Precision weighs Puerarin reference substance, with flowing phased soln, and is configured to the molten of 100 μ g/ml
Liquid, as External standards solutions.
Measuring method：5 bags of gained Puerarin nano liposome preparations in above-described embodiment are taken, content is poured out, mixing is accurate
The sample for being approximately equivalent to Puerarin 10mg is weighed to 100ml measuring bottles, adds flowing phased soln and is diluted to scale.Filtering is drawn continuous
In 20 μ l injections HPLC of filtrate, chromatogram is recorded, the content of Puerarin in this product is calculated by external standard method.Every bag of Puerarin of this product contains
Amount should be the 90%~110% of labelled amount.
The content results for making three batches of samples by oneself are as follows：
The experimental study of pharmacodynamics
Experimental example 1
The oxidativestress damage model of vascular endothelial cell is established in this research using oxidants hydrogen peroxide induction, inquires into Pueraria lobota
Protective effect and mechanism of the root element to Oxidation Induced Damages of Vascular Endothelial Cells, for Puerarin Study on antioxidation provide it is theoretical according to
According to.
1), materials and methods
Key instrument and reagent D MEM culture solutions, fetal calf serum (Gibc companies, the U.S.)；Malonaldehyde (MDA), superoxides
Mutase (SOD) kit, plasminogen activator inhibitor -1 (PAI-1), activator of plasminogen (t-PA) kit (south
Bioengineering Research Institute is built up in capital)；Hydrogen peroxide, Methyl thiazoly tetrazolium assay (MTT), dimethyl sulfoxide (DMSO) (DMSO) (U.S. Sigma
Company)；Annexin V PI staining kits (Roche companies of the U.S.)；Puerarin bulk pharmaceutical chemicals (Zhenping Pharmaceutical Factory).People's navel
Venous endothelial cell (HUVECs) is provided by Tongji Medical Institute's biomedicine experiment center.Microplate reader (Beckman companies of the U.S.)；
Flow cytometer (Becton-Dickinson companies of the U.S.).
Model foundation is grouped with experiment：After the HUVECs frozen is recovered, with the DMEM of the fetal calf serum containing 100ml/L 37
DEG C, cultivate under the saturated humidity of 50ml/L CO2 and 950ml/L air.By the cell of exponential phase 2.5g/L trypsase
It digests and centrifuges, cell is inoculated in 96 well culture plates by the even density of 5 × 103/ml.
Experiment packet is as follows：Normal group is not added with any intervention factor；Hydrogen peroxide group was added in cell culture system
Hydrogen oxide, the final concentration of 0.5mmol/L of hydrogen peroxide；Puerarin group is that Puerarin intervention, Pueraria lobota is added in cell culture system
Root element final concentration is respectively 25,50,100,200mg/L, the hydrogen peroxide effect of same concentration is added after 30min.Each group culture
It carries out following tests afterwards for 24 hours and carries out corresponding Indexs measure, each experimental group is 6 multiple holes.
1. mtt assay detects cell Proliferation：After processing, 10 μ l of MTT (5g/L) are added in each group cell, 37 DEG C be incubated after 4h from
Supernatant is removed after the heart, and 150 μ l of DMSO are added and vibrate 5min, so that crystal is dissolved, merging microplate reader detects suction at 570nm
Luminosity (A).
2. t-PA and PAI Activity determinations：It is quantitative determined in culture solution using Enzyme-linked Immunosorbent Assay double antibody sandwich method principle
T-PA activity and PAI-1 activity.
3. cellular oxidation and antioxidant levels detection：It collects each group cell and carries out ultrasonication, in 4 DEG C, 14000r/min
Under the conditions of centrifuge 20min, take supernatant, with SOD activity and MDA content kit measurements, operated as directed, measure SOD
Activity and MDA content.
4. apoptosis rate：Apoptosis is detected by Annexin V-FITC/PI kit operating instructions.With 2.5g/L's
Trypsin digestion each group cell, the PBS buffer solution being pre-chilled respectively with 4 DEG C are washed 2 times, are removed after 1000r/min centrifugations 5min
Cell is resuspended with combination buffer in cell fragment, and it is about 10 × 106/ml to adjust its cell number.Each sample takes cell suspension
5 μ l and PI (20mg/L) 10 μ l of Annexin V-FITC are added in 5ml streaming pipes in 100 μ L, and room temperature is protected from light incubation after mixing
15min, using flow cytometry analysis each group apoptosis rate.
Statistical analysis is analyzed using SPSS l3.0 statistical packages, and institute's measured value is come with mean ± standard deviation (± s)
It indicates, using one-way analysis of variance, compares two-by-two between q check row groups, indicate that difference is statistically significant with P ＜ 0.05.
2), result
HUVECs is proliferated and the active change MTT method testing results of t-PA, PAI are shown, compared with normal group, peroxidating
HUVECs proliferation activities are substantially reduced (P ＜ 0.01) after hydrogen induction；It can be improved in various degree using the Puerarin of various concentration
Its proliferation activity.As a concentration of 100mg/L of Puerarin, HUVECs proliferation activities significantly improve (P ＜ 0.01), but with dense
Degree increases, and this effect does not increase further.Damage criterion using t-PA and PAI activity as HUVECs is tested, and it is normal
Group compares, and the PAI activity of HUVECs is significantly raised (P ＜ 0.01) after hydrogen peroxide-induced, and t-PA activity is substantially reduced (P ＜
0.01).Compared with hydrogen peroxide group, using, as concentration increases, t-PA activity also gradually increases, and the work of Pal after Puerarin
Property continuously decreases.As a concentration of 100mg/L of Puerarin, effect is most strong (P ＜ 0.01).It is thus determined that follow-up test uses
The Puerarin of 100mg/L carries out.
Puerarin is on the HUVECs of hydrogen peroxide-induced proliferation and the active influence of t-PA, PAI
Influence of the Puerarin to HUVECs oxidative stress：Experiment is using SOD activity and MDA contents as Testing index.
Compared with normal group, the MDA contents of the HUVECs of hydrogen peroxide-induced are significantly raised (P ＜ 0.01), and SOD activity is substantially reduced (P
＜ 0.01)；And SOD activity (P ＜ 0.05) can be significantly improved using Puerarin, reduce MDA contents (P ＜ 0.01).
The apoptosis rate [(40.89 ± 5.26) %] of influence hydrogen peroxide-induced of the Puerarin to HUVECs apoptosis is notable
Higher than control group [(3.54 ± 0.98) %, P ＜ 0.01]；And after Puerarin pre-processes, apoptosis rate be decreased obviously [(15.36 ±
3.14) %, P ＜ 0.01].
3) it, discusses
This result of study finds that Puerarin can obviously reverse the t-PA activity caused by hydrogen peroxide to decline and PAI activity
Increase, prompts it that there is protective effect to the activity of HUVECs.MTT detection methods are by reflecting that the energetic supersession of cell detects cell
Proliferation activity, this experiment find hydrogen peroxide-induced after vast as the open sea HUVECs proliferation activities be decreased obviously, be added Puerarin
It is capable of the decline of apparent reverse both proliferation activity.This research selects MDA and SOD as cellular oxidation and Antioxidant Indexes, sees
Antioxidation of the Puerarin to HUVECs is examined, as a result shows that MDA contents obviously increase when oxidative damage occurs for cell, SOD lives
Property is decreased obviously；And Puerarin reduces MDA contents to the degree of protection of HUVECs with it, improving the active variation tendencies of SOD is
Consistent.This research is to further understand and evaluate the oxidation resistant in vivo studies research of Puerarin to lay a good foundation.
Experimental example 2
This experiment carries out the oral administration biaavailability of gained Puerarin nano liposome preparations in above-described embodiment, investigates
Pharmacokinetics of the Puerarin nano liposomes in beagle dog bodies have investigated the oral bio profit relative to Puerarin suspension
Expenditure.
1), test apparatus and method
Material and instrument：Puerarin reference substance (Nat'l Pharmaceutical ＆ Biological Products Control Institute)；Puerarin bulk pharmaceutical chemicals (Shaanxi Zhenping
Pharmaceutical factory)；Methanol (MERCK), mobile phase are distilled water (self-control) with water；Remaining reagent is that analysis is pure.Agilent1260 high
Effect liquid phase chromatogram instrument (U.S.)；Electronic analytical balance (Ao Haosi)；BT-2000 nitrogen dries up instrument, and (Beijing all directions century science and technology has
Limit company)；Micro-whirlpool mixed instrument (Shanghai Hu Xi analytical instrument factory)；XPN-100 types high speed freezing centrifuge (U.S.'s Beckman
Company) etc..
Chromatographic condition：Using Thermo Zorbax C18 chromatographic columns (5 μm, 4.6mm × 250mm)；Mobile phase is methanol-
3.5mmol/L phosphate buffers (pH 7.8) (15：85)；Detection wavelength 250nm；30 DEG C of column temperature；Flow velocity 1.0ml/min；Into
20 μ l of sample amount.
Experimental design and blood specimen collection：6 beagle dogs (male, weigh about 13kg) are randomly divided into A, two groups (every group 3 of B
Only), it is designed using binary cycle own control, cross-over experiment.Period 1 A group takes orally Puerarin aqueous suspension, and B groups take orally same agent
Amount self-control Puerarin Liposomal formulation, after being spaced the 7d cleaning phases, two groups of exchange medication kinds carry out second round experiment.beagle
After dog overnight fasting, Puerarin aqueous suspension or self-control Puerarin Liposomal formulation are given the next morning on an empty stomach, respectively at administration
Afterwards 1,2,3,4,5,6,8,10,12, whole blood 3mL is taken by stock small saphenous vein for 24 hours, is set in the processed centrifuge tube of heparin sodium,
3500r/min centrifuges 10min, and separated plasma is stored in -20 DEG C of refrigerator, spare.
Sample treatment takes the plasma sample of -20 DEG C of storages, water shower that its quick-thawing, precision is made to draw 0.5ml with measuring,
It sets in tip centrifuge tube, methanol 2ml is added, vortex mixing 2min, 4000r/min centrifugation 10min takes supernatant, nitrogen evaporator 45
DEG C drying, residue be added 0.5ml mobile phases be vortexed dissolving, 18000r/min centrifuge 15min, by above-mentioned chromatographic condition injection HPLC
It measures.
Data processing is handled blood concentration-time data using DAS2.0 softwares, obtains main medicine for power
Learn parameter.Make by oneself Puerarin Liposomal formulation (B) relative to Puerarin suspension (A) bioavilability F=AUC0- ∞ (B)/
AUC0-∞(A).To experiment gained AUC0-t, AUC0- ∞ and Cmax etc. with SPSS softwares carry out preparation between, week during and it is a
Three-factor analysis of variance between body, wherein to AUC0-t, AUC0- ∞.With Cmax elder generations Logarithm conversion, then above-mentioned analysis is carried out, and done
Two one-sided t tests (level of significance α=0.05) finally calculate the credibility intervals % (1-2 α), and nonparametric method is used to tmax
(Wilcoxon signed rank tests) is counted, and carries out equivalence judgement.
2), result
Puerarin suspension is taken orally to beagle dogs and makes the main pharmacokinetic parameters AUC0- of Puerarin Liposomal formulation by oneself
T, AUC0- ∞, Cmax carry out variance analysis, the significant difference of AUC and Cmax of two kinds of preparations after Logarithm conversion.Experiment
The result shows that reaching Cmax up to Cmax Cmax for oral Puerarin suspension after Oral Administration in Rats self-control Puerarin Liposomal formulation
18.2 times, relative bioavailability 1560.6%.
3) it, discusses
Show Puerarin Liposomal formulation compared with Puerarin suspension by the pharmacokinetic parameters of two kinds of preparations, Puerarin
AUC0- ∞ have significant difference (P<0.05), and Cmax/AUC0- ∞ illustrate Puerarin liposome is made without significant difference
Influence of the preparation to Puerarin only increases its degree of absorption, influences very little to its infiltration rate.This experimental result shows pueraria lobata
Plain Liposomal formulation is remarkably improved Pueraria lobota with the Puerarin biology inequivalence in Puerarin suspension, Puerarin after liposome is made
The oral administration biaavailability of root element.
Experimental example 3
The Puerarin Liposomal formulation that this experimental study is prepared according to above-described embodiment is to rat fat and blood viscosity
Influence, to confirm the Puerarin Liposomal formulation prepared according to above-described embodiment with apparent reducing blood lipid and blood viscosity lowering
Effect.
1), materials and methods
Animal and grouping：Choose SD rats 30, half male and half female, 220~250g of weight, balanced diet after a week by its with
Machine is divided into three groups, every group 10, respectively Normal group (A), high lipid food group (B), high lipid food Puerarin group (C).Its
0.9% sodium chloride injection (4ml/kg/d) is fed and be injected intraperitoneally to middle A groups basal feed, and B group high lipid foods are fed and abdominal cavity
0.9% sodium chloride injection (4ml/kg/d) is injected, C group high lipid foods feed and Puerarin suspension (Puerarin is injected intraperitoneally
Raw material is suspended in 0.9% sodium chloride injection, 50mg/kg/d).Three groups of rat feeding environment are consistent, routinely drink water, continuously give
Medicine 30 days.
The acquisition of sample：Sampling evening before yesterday Rat Fast can't help water；After being anesthetized with ether rat, taken using heart extracting blood mode
Blood is divided into two parts, is respectively used to the detection of blood viscosity and blood lipids index.
Detection method：Total cholesterol, triglycerides, high-density lipoprotein in blood plasma are measured using automatic clinical chemistry analyzer
The content of cholesterol, low density lipoprotein cholesterol.Its whole blood viscosity (undercut) is measured using automatic blood rheology detector
And plasma viscosity.
Statistical procedures：Statistical method carries out significance analysis between group using two groups of t inspections, and P ＜ 0.05 have for difference
Statistical significance.
2), result
Influence of the Puerarin to rat fat：Lipids detection result see the table below, and as seen from table, compared with A groups, connect to rat
Continuous high lipid food of raising is after 30 days, and total cholesterol, triglycerides and low density lipoprotein cholesterol content obviously rise in rat plasma
Height, high-density lipoprotein cholesterol content decline, and show that the hyperlipemia model of rat in experiment is successfully established.Compared with B groups,
Rat gives Puerarin dilution (Puerarin 50mg/kg/d) in intraperitoneal injection) after 30 days, total cholesterol, glycerine in blood plasma
Three esters and low density lipoprotein cholesterol content reduce, and wherein plasma total cholesterol levels are decreased obviously, high-density lipoprotein
Cholesterol level is significantly raised, and reaches normal level (P ＜ 0.01).
Influence of the Puerarin to rat fat index
Influence blood viscosity testing result of the Puerarin to rat blood viscosity see the table below, as seen from table, compared with A groups,
After 30 days, rat whole blood viscosity (undercut) and plasma viscosity obviously increase (P ＜ 0.01) continuous high lipid food of raising.Compared with B groups,
The whole blood viscosity (undercut) of rat and plasma viscosity are substantially reduced (P ＜ 0.01) in C groups.
Influence of the Puerarin to rat whole blood viscosity
3) it, discusses
Hyperlipidemia is to substantially reduce angiocardiopathy by reducing blood fat the main reason for causing a variety of angiocardiopathies
Generation.The Puerarin Liposomal formulation that this experimental study is shown as above-described embodiment preparation can effectively reduce rat
The content of total cholesterol, makes hdl concentration increase in serum, and low-density lipoprotein content reduces, and prevents low density lipoprotein
Excessive oxidation occurs for albumen, it was confirmed that makees with apparent reducing blood lipid according to Puerarin Liposomal formulation prepared by above-described embodiment
With.In addition, can effectively reduce the viscosity of blood according to Puerarin Liposomal formulation prepared by above-described embodiment, it is high to improve blood
Solidifying state prompts the incidence that can reduce Cardial or cerebral vascular diseases.
Hyperlipidemia has been known as leading to atherosclerosis and the various diseases of cardiovascular and cerebrovascular by world medical circle at present
Primary risk factor easily leads to the generation of atherosclerosis if hyperlipidemia cannot control for a long time.This test result is aobvious
Show has apparent effect for reducing blood fat according to Puerarin Liposomal formulation Puerarin prepared by above-described embodiment.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, various changes and improvements may be made to the invention without departing from the spirit and scope of the present invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent defines.
Claims (6)
1. a kind of Puerarin Liposomal formulation for treating atherosclerosis, it is characterised in that：The preparation contain with
The component of lower mass ratio：1 part of Puerarin, carrier phosphatidase 3~9 part, 1~6 part of surfactant, 3~10 parts of excipient, disintegration
3~10 parts of agent.
2. a kind of Puerarin Liposomal formulation for treating atherosclerosis as described in claim 1, it is characterised in that：
The carrier phosphatide is one kind in soybean lecithin, hydrogenated soya phosphatide, lecithin, hydrolecithin, cephalin.
3. a kind of Puerarin Liposomal formulation for treating atherosclerosis as described in claim 1, it is characterised in that：
The surfactant is one kind in polyethylene glycol -15- hydroxy stearic acid esters, Tween 80, PLURONICS F87.
4. a kind of Puerarin Liposomal formulation for treating atherosclerosis as described in claim 1, it is characterised in that：
The excipient is 30 POVIDONE K 30 BP/USP 25, PVP K30,30 POVIDONE K 30 BP/USP 60,30 POVIDONE K 30 BP/USP 90, polyethylene glycol 2000, polyethylene glycol
4000, one kind in Macrogol 6000, PEG 8000.
5. a kind of Puerarin Liposomal formulation for treating atherosclerosis as described in claim 1, it is characterised in that：
The disintegrant is crosslinked polyvinylpyrrolidone.
6. a kind of preparation method as described in claim 1 for treating the Puerarin Liposomal formulation of atherosclerosis,
It is characterized in that：It comprises the steps of：
1), in mass ratio example take a Puerarin, 3~9 parts of carrier phosphatide, 1~6 part of surfactant, 3~10 parts of excipient and
3~10 portions of disintegrants, which are added to stirring in absolute ethyl alcohol, makes it fully dissolve and be uniformly mixed；
2), mixture is dried under reduced pressure to viscous liquid in stirring condition, disintegrant is added and is uniformly mixed with mixture；
3), continue to be dried under reduced pressure to absolute ethyl alcohol and completely remove totally, the solid matter of acquisition is crushed, is sieved and can obtain
To the Liposomal formulation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106821987A (en) * | 2017-03-16 | 2017-06-13 | 四川大学 | A kind of liposome and preparation method and application for carrying phenolic hydroxy group insoluble drug |
| CN110859869A (en) * | 2019-12-20 | 2020-03-06 | 中国中医科学院中医药科技合作中心 | A composition for preventing and treating cardiovascular and cerebrovascular diseases, and its preparation method |
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| CN101455648A (en) * | 2009-01-05 | 2009-06-17 | 浙江康恩贝制药股份有限公司 | Puerarin freeze-drying preparation |
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2017
- 2017-03-07 CN CN201710131031.4A patent/CN108567741A/en active Pending
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| CN101455648A (en) * | 2009-01-05 | 2009-06-17 | 浙江康恩贝制药股份有限公司 | Puerarin freeze-drying preparation |
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| CHENG-FENGLUO等: "Pharmacokinetics, tissue distribution and relative bioavailability of puerarin solid lipid nanoparticles following oral administration", 《INTERNATIONAL JOURNAL OF PHARMACEUTICS》 * |
| 王婷: "提高葛根素口服生物利用度剂型的研究进展", 《中国当代医药》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106821987A (en) * | 2017-03-16 | 2017-06-13 | 四川大学 | A kind of liposome and preparation method and application for carrying phenolic hydroxy group insoluble drug |
| CN110859869A (en) * | 2019-12-20 | 2020-03-06 | 中国中医科学院中医药科技合作中心 | A composition for preventing and treating cardiovascular and cerebrovascular diseases, and its preparation method |
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