The Study On Nanoemulsion Of Fructus Perillae Oil

In this research,we used the advanced nanotechnology to transform the traditional Chinese herb fructus perillae oil,prepared fructus perillae oil nanoemulsion.Screening the prescription through basic researching of the blank nanoemulsion,used pseudo-triangular phase diagram to optimal prescription and used titrimetric method to prepare fructus perillae oil nanoemulsion,the quality stability,security,pharmacodynamics and pharmacokinetics of fructus perillae oil nanoemulsion were systematic studied.The abstracts are as follows:1.Basic research of blank nanoemulsionTo screen blank nanoemulsion prescription and prepare it,carried on the inspection to its influencing factor.Used the hydrophilic-lipophilic balance and nanoemulsion evaluation criteria to screen prescription;used titrimetric method to prepare blank nanoemulsion;inspected the shape and particle diameter through transmission electron microscope and laser particle size distribution meter;inspected influential factor of nanoemulsion formation though pseudo-triangular phase diagram.The result indicated that nanoemulsion prescription is, the surfactant is the Tween (80 and 60),Span (80 and 60),EL40 and RH40;the cosurfactant is dehydrated alcohol,glycerine,1,2-butylene-glycol and n-butanol;the oil phase is IPM,whiteruss,olive oil and wheat embryo oil;the blank nanoemulsion is clarify,diaphanous,stramineous or light blue opaline liquid,assumed globular below electron microscope,particle diameter is 20.7 nm,the spreading rate of methylene blue is obviously greater than magdala red III;the influencing factor inspection result showed that surfactant and oil phase type and proportion are most main factor;in nanoemulsion form scope,the cosurfactant HLB value is bigger,the chain of cosurfactant is shorter and the HLB value of oil and cosurfactant are better matcher,nanoemulsion area is bigger;in a range,the medicine joins increased nanoemulsion area.The quality of blank nanoemulsion is stable,the type is oil in wate.2. Development and quality evaluate of fructus perillae oil nanoemulsionTo prepare fructus perillae oil nanoemulsion and overall assess it's quality.To establish the analytical method for content determining of fructus perillae oil nanoemulsion drug delivery system,to inspect it's carry dosage and entrapment efficiency;used of transmission electron microscopy,laser particle size distribution device,rotary viscometer and refractometers conductivity,etc.to evaluate the quality;to inspect the stability through determin stability parameter,accelerated test,light accelerated test,classical homeothermia accelerated test and peroxide value determination.Gas chromatography detection results showed that the good linear range of the preparation was 50 ng/mL ~1 600 ng/mL,the mean recovery is 99.3 percent, RSD of the recovery is 0.69 percent,the mean retention time is 33.398 min,the type duplication is 0.52 percent and 0.34 percent;the good linear range of the blood plasma was 10 ng/mL~1 600 ng/mL,the mean recovery is 95.8 percent,RSD of the recovery is 2.95 percent,the mean retention time is 33.545 min,the type duplication is 0.58 percent and 0.83 percent;quality evaluate results show that the nanoemulsion drug delivery system of fructus perillae oil contains RH40/ethyl alcohol/wheat embryo oil is transparent spherical bubble,its average diameter is 26.0 nm,encapsulation efficiency is over 80 percent,consistency is 5.64±0.09 mm2.s-1,refractive index is 1.178±0.0023 nD20,conductivity is 366±1.36 mS-1,Zeta electric potential is -2.18±0.04 mV.The stability parameter of fructus perillae oil nanoemulsion is 4.60±0.16.The analysis method we established in this study have high recovery,accuracy and repeatable,it can be used in quality control and dynamics research. Fructus perillae oil nanoemulsion drug delivery system is stable, correspond to medication requirement.3. The safety evaluation on fructus perillae oil nanoemulsionThrough quadriceps injection testing, hemolytic test, vascular stimulation test, pyrogen testing, mouse acute toxicity and cytotoxicity test to estimate the safety of fructus perillae oil nanoemulsion. Quadriceps injection testing showed that the injection site has lightly congestion,it's diameter is below 0.5 cm,belongs to 1 class;hemolytic test showed no clarity red in tube,and RBC are all sink,the upper liquid clarity,the red blood cells can dispersed after rock and fructus perillae oil nanoemulsion without hemolysis;vascular stimulation test results showed that the organizational structure of normal skin,skin thickening no congestive,subcutaneous tissue has no edema and other morphological changes,ear vein has no expansion,artery wall has no thicker,pyrogen test results showed that body temperature increase of every rabbit is lower than 0.6℃,the sum of three rabbits temperature increase is lower than 1.4℃;acute toxicity results showed that the maximal tolerance dose test on mice of the fructus perillae oil nanoemulsion is 140 g/kg,converted into adult's dosage (50 kg) is 3 5000 g/kg,7 777 times of clinical amount used,calculated its accumulation LD50 is 602.0 g/kg; cytotoxicity test showed that compared with normal cell,fructus perillae oil nanoemulsion group cell growth velocity is slightly step down,the shape has no significant difference,belongs to 1 level.Fructus perillae oil nanoemulsion has no vascular irritation,has no hemolytic crisis, pyrogen inspection conforms to the regulation,has no evident toxic effect to L cell,and has high security.4. Study of pharmacodynamics on fructus perillae oil nanoemulsionThrough against acute hyperlipemia,the impact of hypertension rats blood pressure and heart rate and induction EMT-6 cell apoptosis test to study the pharmacodynamics.Acute hyperlipemia test showed injection of 0.25 mL/10 g 75 percent of the yolk milk can be successfully reproduced in mice with acute high blood lipid model,the relative magnitude of reduce mouse plasma total cholesterol,triglyeride and atherogenic index is 4.48 times,2.06 times and 2.61 times than fructus perillae oil capsulae,and 4.62 times,1.28 times and 1.45 times than fenofibrate;hypertension test showsed it can significantly reduce blood pressure,compared with the model group,the blood pressure decreased 28 percent (P<0.01),heart rate increase 14 percent(P<0.01),and cardiac index reduced 19 percent (P<0.01);apoptosis test result showed that fructus perillae oil nanoemulsion has significantly inhibited proliferation when drug concentration is 100μg/mL~800μg/mL,apoptosis of fructus perillae oil nanoemulsion is better than fructus perillae oil capsule,significant differences on statistically (P<0.05).Fructus perillae oil nanoemulsion of 800μg/mL dose induced EMT-6 cells,under TEM showed cytoplasmic margination,apoptotic bodies formed within the cytoplasmic vacuoles,and after 48 h,we can see the length of a DNA degradation of 180~200 bp integer multiple oligonucleotide fragments of the ladder.After 24 h,apoptosis peak apparence and peak extension with the processing time increased by flow cytometer detection. Fructus perillae oil nanoemulsion can significantly cut down mouse triglyeride,total cholesterol and low-density lipoprotein,and raise high-density lipoprotein,fructus perillae oil nanoemulsion can significantly cut down hypertension rat blood pressure,fructus perillae oil nanoemulsion has evident apoptosis to EMT-6 cell.In short,the pharmacodynamic of fructus perillae oil nanoemulsion is much better than fructus perillae oil capsule.5. The pharmacokinetics study on fructus perillae oil nanoemulsionTo study the fructus perillae oil nanoemulsion on pharmacokinetics.The fructus perillae oil nanoemulsion was administered to the rabbit by lavage with 25 mL/kg,in the time of 0.5h,1h,1.5h,2h,2.5h,3h,3.5h,4h,8h,10h,12h,14h,18h,24 h,α-LNA was determined through gas chromatographyin,the medicine kinematics parameter was calculated.The result showed that fructus perillae oil nanoemulsion is fit for the one room open model in rabbit.The average peak time values of fructus perillae oil nanoemulsion was 4.929 h,extend 1.650 h than capsule (3.279 h);the average highest plasma concentration Cmax(136.058μg/L) is 1.2 times of capsule (110.237μg/L),the relative bioavailability is 283.1% to capsule pharmacokinetics.The relative bioavailability of fructus perillae oil nanoemulsion increased significantly than fructus perillae oil capsulae,and has a certain degree of sustained-release effect.Fructus perillae oil nanoemulsion has stable quality,high safety,pharmacodynamic action has significant raised than tradition dosage form of fructus perillae oil.The research outcome has applied for national invention patent,apply order is 200610104621.X.