| Objective:1.Preparation of icariside II(ICSII)new nano-dosage.2.To establish the high performance liquid chromatography mass spectrometry(LC-MS)method to determined ICSII concerntration in plasma and tissue of rats.3.To determin the pharmacokinetic parameters and evaluate the tissue distribution properties of the nano-liposomes of ICSII in rats.Methods:1.Sephadex LH-20 microcolumn centrifuge-HPLC method was established to determinate the entrapment efficiency of nano-liposomes.The applicable methods to determinate the entrapment efficiency of the liposomes were screened out by interfering the centrifugal frequency and free drug concentration.After the absorption of Sephadex LH-20 microcolumn centrifuge,liposomes were centrifuged and eluted by a RCF(relative centrifugal force)of 45 g with PBS.From the methodological observation,the method showed a good specificity,a good linear relationship in the range of 0.01-0.35 mg/m L and the intra-and inter-day precision were satisfactory(RSD<2.00%),and the recovery of free drug was 98.86%.ICSII liposomes were prepared by film dispersion-ultrasonic method and the preparation technology was preliminarily optimized by single factor.2.LC-MS was used to determinate the ICSII liposomes concentration in plasma of rats.Samples were extracted by ethyl acetate,and then they were blow-dried at room temperature.Thereafter,samples were-dissoluted with methyl alcohol then loaded samples.ICSⅠ was used as internal standard.Chromatographic condition was listed as follows: gemini-Nx3μC18110A(150×3.0 mm,3 μm),phenomenex chromatographic column,the mobile phases was 0.25% formic acid aqueous solution and 0.25% formic acid acetonitrile solution,flow rate was 0.5 m L/min,which were used for gradient elution.ESI(Electrospray Ionization),positive ion mode and MRM(Multiple Reaction Monitoring)were taken to scan,and the quantitative ion pair is composed by m/z 515.2/313.2(ICSII)and m/z 531.2/313.2(ICSI).The linear range in plasma of ICSII was listed as follows: 3.80-760.00 ng/m L(R2>0.99),the intra-and inter-day precision(RSD<8.39%),degree of accuracy(±6.91%),extraction recovery: 84.98%-98.55%(RSD<8.44%).The method is proved to be rapid,sensitive and higher accuracy.Male SD rats randomly divided into two groups including a single dose of ICSII(iv.5 mg/kg)group and ICSII nano-liposomes group.ICSII or ICSII nano-liposomes group were detected by LC-MS at different time pointsand then calculated pharmacokinetic parameters by DAS3.2.8 software.3.As the same as the processing method of plasma sample,LC-MS method was established to determinate the ICSII of tissue from rats.The linear range in plasma of ICSII was listed as follows: 3.83-766.00 ng/mL(r2>0.99),the intra-and inter-day precision(RSD<9.75%),degree of accuracy(±9.31%),extraction recovery: 84.95%-97.60%(RSD<10.05%).Seventy-two SD rats randomly divided into twelve groups and treatment with ICSII suspension and ICSII nano-liposomes by tail intravenous injection.Thereafter rats were killed by dislocated method,and the tissues of brain,heart,liver,spleen,lung,kidney were collected from rats.The concerntration of ICSII were detected by LC-MS and then calculated pharmacokinetic parameters by DAS 3.2.8 software.Results:1.The optimal preparation technology of ICSII liposomes was listed as follows: soybean lecithin:cholesterol=1:20:5,methanol 0.5 mL,chloroform 6m L,PBS 3 mL,ultrasonic 30 min,EE% gained 95.6.PDI was 0.272,and transmission electron microscope observed the outline of ICSII nano-liposomes were spherical or nearly spherical.2.DAS3.2.8 was used to calculate the pharmacokinetic parameters of ICSII after the rats were treated with ICSII nano-liposomes or ICSII.The t1/2z in rats of the control group was 1.81±0.67 h,the nano-lipsome group was 3.25±1.49 h(p < 0.05).The MRT(0-∞)and MRT(0-t)of the control group was 0.45±0.39 h and 0.49±0.48 h,the nano-lipsome group was 1.60±0.65 h(p<0.01)and 2.22±1.03 h(p<0.01),The AUMC(0-∞)and AUMC(0-t)of the control group was 575.14±135.47 h×h×μg/L and 608.96±181.33 h×h×μg/L,the nano-lipsome group was 1328.73±692.10 h×h×μg/L(p<0.05)and 2059.45±1478.74 h×h×μg/L(p<0.05).3.DAS3.2.8 was also used to calculate the pharmacokinetic parameters of ICSI or ICSII nano-liposomes in main organs of rats.The t1/2z in rats heart of the control group was 0.80±0.30 h,the nano-lipsome group was 1.21±0.29 h(p < 0.05).The MRT(0-∞)in rats’ spleen of the control group was 0.34±0.31 h,the nano-lipsome group was 1.35±1.09 h(p < 0.05).The MRT(0-∞)in the rats’ lung of the ICSII group was 0.87±0.44 h,the nano-lipsome group was 1.42±0.51 h(p < 0.05).The AUMC(0-t)and AUMC(0-∞)in the rats’ spleen of the control group was 128.36±68.17 h×h×μg/L and 149.85±108.93 h×h×μg/L,the group of nano-lipsome was 357.22±258.46 h×h×μg/L and 383.09±263.66 h×h×μg/L(p <0.05).Conclusion: ICSII nano-liposomes were prepared,the entrapment efficiency is more than 90%,and the diameter of particles is less than 100 nm;its dispersibility is very well.A rapid,sensitive and reliable method for the determination of ICSII concentrations in rat plasma and tissues was established.The results of pharmacokinetic experiment shows that the elimination half-life and mean residence time of ICSII was extended after ICSII was prepared for nano-liposomes.In the tissue distribution,the elimination half-life of ICSII in heart and blood were extended after ICSII nano-liposomes were given;the mean residence time of blood,spleen and lung were significantly increased,respectively,and the AUMC of blood and spleen is larger than ICSII group. |
PDF Full Text Request