Studies On Long-circulating Oridonin Liposomes

Oridonin,a complex diterpenoid derivative,is extracted from traditional chinese herbs, Rabdosia rubescens which is one of the most important traditional Chinese herbs commonly used in clinical treatment nowadays.More than half a century ago,oridonin was shown to have a variety of biological effects such as immunoregulatory and anti-inflammatory functions as well as antiviral functions especially in upper respiratory tract infections.Recent laboratory and clinical data suggest that oridonin is a very effective antitumor agent with profound effects on a number of malignant diseases such as prostate,breast,and non-small cell lung cancers.However,oridonin is unstable,easily oxidized and nonhydrophilic. Biological half life of oridonin is short.These disadvantages limited the clinical application of the chemotherapy medicine.In order to improve the detention time and concentration of oridonin in blood,long-circulating oridonin liposomes were prepared.The distribution of long-circulating oridonin liposomes in blood and tissues in mice were studied.The theraputic effects of long-circulating oridonin liposomes on H₂₂ mousehepatoma carcinoma cells induced cancer mice were evaluted.The extraction and purification processes of oridonin from Rabdosia rubescens were first studied.Rabdosia rubescens powder was ultrasonic extracted with 95%ethanol with a 12:1 ratio of solvent:Rabdosia rubescens for 30 minutes.Repeat the ultrasonic extraction for another 2 times.The combined ethanol extract was then ultrasonic decolorized with active carbon for 20 minutes.Repeat the ultrasonic decolorization one more time.The dosage of active carbon was 0.24g for 1g of Rabdosia rubescens used.The preparative high performance liquid chromatography separative conditions of crude oridonin were investigated. The decolorized oridonin was separated by a reverse phaseμBondapack^TMC₁₈ column(80mm i.d×300mm,grain size 25μm～40μm).The temperature of the column was maintained at 40℃.The mobile phase was CH₃OH-H₂O(50/50,V/V).The flow rate was 60mL·min^-1.The detective wave-length was set to 238nm and the injective volumn was 10mL.The eluted solution of 16min～23min was evaporated and vacuum dried.White powder of oridonin was obtained with 99.0%by HPLC.The extraction yield of oridonin was 65.4%based on analysed concentration of oridonin in Rabdosia rubescens.The structure of the final product separated was identified by LC-Ms,elemental analysis,UV,IR,HPLC and NMR spectrum.To optimize preparing technique of long-circulating oridonin liposomes and establish HPLC method for determination of oridonin liposomes,the influence of various factors, including the dosage of lecithin,cholesterol,oridonin,PEG₂₀₀₀-DSPE,hydrous medium and kinds of hydrous medium on the entrapment efficiency and ratio of loading drug were investigated,and the optimum formula was selected through orthogonal design test.The concentration of oridonin was determined by HPLC using a reverse phase Lichrospher C₁₈ column(4.6mm i.d×250mm,grain size 5μm).The temperature of column was maintained at 40℃.The mobile phase was CH₃OH-H₂O(70/30,V/V).The flow rate was 1mL·min^-1.The detective wave-length was set to 238nm and the injective volumn was 10μL.In the optimal process,0.26g lecithin,0.14g cholesterol,0.5g PEG₂₀₀₀-DSPE,0.04g oridonin and 30mL pure water was used.The product had good size distribution from 100nm to 200nm.The mean particle size diameter was 150nm,entrapment efficiency was 95.4%and ratio of loading drug was 4.24%.The release time of oridonin from liposome was 96h.The preparative process of oridonin liposomes is feasible.The HPLC method is simple,rapid and the results is accurate and reliable.The regular oridonin liposomes was prepared in the same way without adding polyethylene glycol-distearoylphosphatidylethanolamine(PEG₂₀₀₀-DSPE).The long-circulating oridonin liposomes and regular oridonin liposomes were stored at 5℃for 80 days without visible change.Entrapment efficiency had no change.The result indicated that long-circulating oridonin liposomes and regular oridonin liposomes were stable at 5℃.To study the biodistribution and pharmacokinetics of long-circulating oridonin liposomes and regular oridonin liposomes in mice,HPLC method was developed for the determination of the contents of oridonin in blood and tissues in mice.The long-circulating oridonin liposomes,regular oridonin liposomes and free oridonin were tail iv administrated and results were compared.The relative targeting efficiency of long-circulating oridonin liposomes in the liver,spleen,lung,heart and kidney were 2.10,1.95,1.34,2.39 and 0.84;The relative targeting efficiency of regular oridonin liposomes in the liver,spleen,lung,heart and kidney were 4.47,3.84,1.49,1.89 and 0.92.The concentration-time curves of long-circulating oridonin liposomes was fitted to the two-compartment model with T_1/2^α＝3.18h,T_1/2^β＝98.36h, AUC_0-48h＝888.91 h·μg.mL^-1,V_c＝19.17mL·Kg^-1;The concentration-time curves of regular oridonin liposomes was fitted to the two-compartment model with T_1/2^α＝2.54h,T_1/2^β＝33.98h, AUC_0-48h＝356.69 h·μg·mL^-1,V_c＝20.01mL·Kg^-1;The concentration-time curves of free oridonin was fitted to the two-compartment model with T_1/2^α＝0.63h,T_1/2^β＝6.19h, AUC_0-48h＝156.07 h·μg·mL^-1,V_c＝10.66mL·Kg^-1.Long-circulating oridonin liposomes was found to reach a long circulation time.For evaluation of the effect of long-circulating oridonin liposomes,regular oridonin liposomes and free oridonin on tumor cells in vivo,H₂₂ mousehepatoma carcinoma cells were transplanted subcutaneouly in mice to induce growth of solid tumors.Tumor weight inhibition rate was then detected after administration of the oridonin preparations. Long-circulating oridonin liposomes,regular oridonin liposomes and free oridonin all showed antitumor effect.The long-circulating oridonin liposomes showed stronger antitumor effect than regular oridonin liposomes and free oridonin.The inhibitory rate of long-circulating oridonin liposomes,regular oridonin liposomes and free oridonin was 85.4%, 71.4%,63.7%at a dose of 3.35×10^-2 g·Kg^-1·d^-1,respectively.