The Research For The Preparation Of Nanostructured Lipid Carrier System Of The Volatile Oil From Folium Atremisiae Argyi And Its Anti-hbv Activity

Folium Atremisiae Argyi is the dry leaf of Arterial argyi Lévl.et Vant.,which is the chinese traditional medicine.The volatile oil from Folium Atremisiae Argyi are the main active constituents of the leaves.It has anti-bacterial,anti-virus,relieve asthma,antitussive,expectorant,anti-allergic,immune,protect liver and gallbladder,and so on.In the early stage of this study,we first founded that the volatile oil of the leaves was resistant to hepatitis B virus.But taking into account the volatile oil is water insoluble,volatile,unstable,affecting the efficacy.Therefore,nanostructured lipid carrier of volatile oil from Folium Atremisiae Argyi was studied in this paper,including the preparation,quality evaluation,pharmacokinetics,tissue distribution and pharmacodynamics.This study has not been reported.1.Preparation and quality evaluation of nanostructured lipid carrier of volatile oil from Folium Atremisiae ArgyiThe NLC of volatile oil from Artemisia argyi was prepared by the method of heated melting and ultrasonic dispersion,and the preparation process and prescrip-tion were screened by single factor experiment and orthogonal test.The optimum preparation process was as follows: the colostrum was stirred for 10 min,the ultrasonic time was 15 min,and the ultrasonic amplitude was 100%.Prescription:Poloxamer 188 was 0.45 g,egg yolk lecithin was 0.15 g,glyceryl monostearate was0.1g,octanoic acid / capric triglyceride was 0.1g,volatile oil was 0.1g,distilled water was added to 20 m L.The method for determination of main components content was established by gas chromatography.The method has the advantages of high precision and good reproducibility,and the recoveries of eucalyptus,camphor and borneol are between95% and 105%.The contents of eucalyptol,camphor and borneol were 16.34%,3.32% and 5.92% respectively.Particle size and zeta potential were determined by nano-particle size analyzer.The overall quality and process was evaluated by the fingerprint.Entrapment efficiency,drug loading,in vitro release and stability was investigated to evaluate their physicochemical properties.The average particle size was（72.33 ± 1.93）nm,PDI was（0.273 ± 0.0045）,and the Zeta potential was（-30.59 ± 1.42）mv.The encapsulation efficiency drug loading were 87.49%,86.45%,92.12%.drug loading were8.25%,2.00%,3.38%.The results of fingerprint show that the similarity of 10 different batches of preparations is more than 0.990,indicating that the consistency of 10 different batches of preparations is good and process quality is stable.In vitro release experiment showed that the release rate of NLC was 61.12%and the original drug was 72.46% at 120 h.the release rate of the NLC at the same time was lower than that of the original drug.The results showed that the volatile oil NLC had sustained release effect.The stability results showed that the NLC of volatile oil was easy to change under high temperature and strong light conditions,should be placed under the conditions of 4 ℃ away from light.2.Study on pharmacokinetics and tissue distribution of the NLC and volatile oilIn this paper,the method of GC determination of eucalyptol in the plasma was established for the first time,and the pharmacokinetics of eucalyptol in the rat was studied.Oral dose of volatile oil was 1000mg/kg,tail vein injection dose was 140mg/kg,the NLC of volatile oil in the same dose of volatile oil,at different time points to draw blood after administration,determinate the blood concentration of eucalyptol by GC Method,and the DAS3.0 software was used to process the data,then the pharmacokinetic parameters were obtained.After 4h,the maximum blood concentra-tion of NLC was 11.4835μg/L,the half-life was 8.727 h,and the maximum plasma concentration of volatile oil was 9.1064μg/L and the half-life period was 7.779 h.the AUC_0-∞of the NLC was 103.281 μg/L?h,and the AUC_0-∞of the volatile oil was160.283μg/L?h.The results showed that the NLC of volatile oil changed the pharmacokinetic characteristics of volatile oil,prolonged the time of drug in vivo and improved the bioavailability of the drug.In this paper,the distribution of nanoparticles in different tissues at different times was investigated by small animal live imaging.Group was divided into group A,group B and group C,group A was saline group,group B was Di R dye group,group C was Di R-NLC group,was anesthetized at different time points,placed in small animal live imaging instrument to take picture.The tissues of the heart,liver,spleen,lung and kidney were removed from the nude mice and placed in a small animal live imager to observe the distribution of tracer nanoparticles in different tissues of mice.The results showed that the nanoparticles were gradually accumulated in the liver over time,and the fluorescence intensity of the liver in Di R-NLC group was stronger than that in the dye group,and it was founded that the NLC of volatile oil had Liver targeting.3.To study the anti-HBV pharmacodynamics of the NLC of volatile oil from Folium Atremisiae ArgyiThe duck hepatitis B virus model was used to observe the anti-HBV effect of volatile oil and the NLC.1 day old ducks were fed for 3 days and then injected with strong positive serum containing DHBV.After 7 days,the ducks infected with hepatitis B virus were screened by PCR.Then,112 positive ducks were randomly divided into 8 groups,given different doses of volatile oil and NLC,continuous administration for 15 days,The DHBV levels in serum and liver tissue were measured at 5 days,10 days,15 days after administration and 3 days after drug withdrawal,the anti-hepatitis B virus activity of volatile oil and NLC was evaluated.The results showed that volatile oil and NLC had inhibitory effect on hepatitis B virus in duck serum and liver tissue,and the inhibitory effect was more obvious at high dose,and the inhibitory effect of the NLC of volatile oil was more significant than volatile oil.