| Objective:In this study,liposomes were used as the model membrane of biomembrane.Paeonol,apocynin and 2'-hydroxy-5'-methoxy acetophenone(HMA)were used to prepare DPPC liposomes.By studying and comparing the encapsulation efficiency,stability of liposomes and the interaction between three kinds of drug molecules and liposome bilayer membrane,the effects of drugs on the stability,phase and location of liposomes were discussed to explore the possible mechanism of action of drugs in the body.Methods:1.To establish a HPLC method for the determination of paeonol,apocynin and HMA,including the experiments of specificity,linearity,repeatability,precision,stability,and accuracy.2.The entrapment efficiency of paeonol,apocynin and HMA/DPPC liposomes with different molar concentrations at 0,3,7,15 and 30 days were determined by ultra-high-speed centrifugation and HPLC.The leakage rate of liposomes under different storage conditions was obtained.At the same time,the leakage rate of liposomes in artificial gastric liquid and artificial intestinal liquid water bath for 0.5 h,1 h,2 h and 4 h was determined and the effect of drug concentration on the stability of liposomes was investigated.3.The interaction of paeonol,apocynin and HMA with liposome membrane was studied by differential scanning calorimetry(DSC).The effects of drug addiction on the thermodynamic properties of liposomes were studied by phase transition temperature and half peak width.The formation of lamellar phase and the phase state of liposomes at different temperature were determined by synchrotron radiation X-ray diffraction(SXRD)to study the effect of drug molecules with different mole percentages on the phase state of liposomes.Fourier transform infrared spectroscopy(FTIR)was used to study the effects of three kinds of drug molecules with different molar percentages on the peak positions of characteristic functional groups(CH3,CH2,C=O,PO2-)and the area percentage of hydrated C=O of DPPC,and the effects of drug molecules with different mole percentages on the molecular level of DPPC.4.Comparing the effects of paeonol,apocynin and HMA with DPPC liposomes,and to explore the possible reasons for the differences in their structures and pharmacological effects.Results:1.ACE EXCEL C18 column(4.6 × 250 mm,5 ?m)was used;methanol:water(60:40)was used as mobile phase;flow rate was 1.0ml·min-1;detection wavelength was 274 nm;column temperature was 25?;injection volume was 10 ?L.The results showed that DPPC liposomes did not interfere with the detection of the three drugs.Paeonol had a good linear relationship in the range of 0.27?8.10 ?g,and the linear regression equation was Y=3115.7X-90.51(R2=0.9999).The RSD of intraday and inter day was 0.29%(n=6)and 0.07%(n=3),respectively,with good precision.The RSD of repeatability was 0.10%(n=6),with good repeatability.The RSD of stability was 0.08%(n=7),and paeonol sample was stable within 12 h.The recoveries of paeonol/DPPC liposomes at low,medium,and high concentrations were 96.17 ± 0.35%,104.60 ±0.37%and 95.88 ±0.15%(n=3),respectively,with RSD of 0.37%,0.36%and 0.15%(n=3),the recovery was good.Apocynin had a good linear relationship in the range of 0.27?8.10 ?g,and the linear regression equation was Y=3553.6X+24.306(R2=1).The RSD of intraday and inter day was 0.14%(n=6)and 0.10%(n=3),respectively,with good precision.The RSD of repeatability was 0.05%(n=6),with good repeatability.The RSD of stability was 0.07%(n=7),with good stability within 12 h.The recoveries of apocynin/DPPC liposomes at low,medium,and high concentrations were 98.41 ± 0.11%,103.01 ± 0.11%and 98.78± 0.04%(n=3),respectively,with RSD of 0.11%,0.11%and 0.04%(n=3),the recovery was good.HMA had a good linear relationship in the range of 0.022?2.20 ?g,and the linear regression equation was Y=119.12X-0.5001(R2=1).The RSD of intraday and inter day was 0.07%(n=6)and 0.04%(n=3),respectively,with the precision was good.The repeatability RSD was 0.11%(n=6),with the repeatability was good.The stability RSD was 0.05%(n=7),the sample of HMA was stable in 12 h.The recoveries of HMA/DPPC liposomes at low,medium,and high concentrations were 97.75±0.88%,95.83±0.17%and 95.80 ± 0.12%(n=3),respectively,with RSD of 0.91%,0.18%and 0.13%(n=3),the recovery was good.2.The encapsulation efficiency of 2.5 mol%,5 mol%,10 mol%,15 mol%,20 mol%and 25 mol%paeonol/DPPC liposomes were 75.73 ± 0.91%,74.87±0.45%,76.42± 0.37%,74.23±0.64%,77.68 ± 0.85%and 73.96 ± 0.36%(n=3),respectively.The leakage rate was the lowest when the paeonol/DPPC liposomes were stored in refrigerator at 4?(dark).The effect of artificial gastric juice on the stability of liposomes was greater than that of artificial intestinal juice.DSC results showed that when the concentration of paeonol was more than 15 mol%,the pre phase transition disappeared,and the main phase transition peak width increased with the addition of paeonol at different mole percentages.When the concentration of paeonol was 2.5 mol%,the pre phase transition and main phase transition temperatures increased,but the main phase transition enthalpy decreased.When x=5 mol%,the main phase transition temperature of paeonol/DPPC liposomes decreased.When 5<x<15 mol%,the main phase transition enthalpy of paeonol/DPPC liposomes increased and the main phase transition temperature changed little.When x>15 mol%,the main phase transition enthalpy and main phase transition temperature of paeonol/DPPC liposomes decreased.SXRD results showed that paeonol/DPPC liposomes with different molar concentrations formed lamellar liposomes,and when x>15 mol%,the liposomes were in undulation gel phase without heating.FTIR results showed that the addition of paeonol had no effect on the characteristic peak position of DPPC.When 0<x?5 mol%,the peak area percentage of hydrated vC=O was basically unchanged.When 5<x?15 mol%,the peak area percentage of hydrated vC=O increased significantly and then decreased,and all of them were greater than 5 mol%.When 15<x?25 mol%,the peak area percentage of hydrated vc=o decreased,with the increase of paeonol concentration,the peak area percentage of hydrated vc=o was almost unchanged.3.The entrapment efficiency of 2.5 mol%,5 mol%,10 mol%,15 mol%,20 mol%and 25 mol%apocynin/DPPC liposomes were 75.3 1 ± 0.73%,74.75± 0.79%,75.59 ± 1.07%,74.11 ±0.92%,77.07 ± 0.62%and 73.77±0.87%(n=3),respectively.The leakage rate was the lowest when stored in refrigerator at 4?(dark).The effect of artificial gastric juice on the stability of liposomes was greater than that of artificial intestinal juice.DSC results showed that when the drug concentration was higher than 2.5 mol%,the pre phase transition of liposomes disappeared,and the half peak width of main phase transition increased and phase separation appeared with the addition of different mole percent concentration of apocynin;with the increase of drug concentration,the main phase transition temperature of liposomes continued to decrease,and the main phase enthalpy fluctuated.The results of SXRD showed that apocynin/DPPC liposomes with different molar concentrations formed lamellae liposomes.When x>10 mol%,the liposomes were in undulation gel phase without heating,and the pretransition temperatures of 5 mol%and 10 mol%of apocynin/DPPC liposomes were 33 and 31 degrees,respectively.FTIR results showed that the addition of different mole percentages of apocynin had no effect on the characteristic peak position of DPPC.When 0<x?2.5 mol%,the peak area percentage of hydrated vC=O increased slightly;when 2.5<x?5 mol%,the peak area percentage of hydrated vC=O decreased significantly;when 5<x?15 mol%,the peak area percentage of hydrated vC=O increased first and then decreased;when 15<x?25 mol%,the peak area percentage of hydrated vc=o decreased and the area percentage of vc=o peak always increased.4.The encapsulation efficiency of 2.5 mol%,5 mol%,10 mol%,15 mol%,20 mol%and 25 mol%HMA/DPPC liposomes were 85.13 ± 1.52%,85.72 ± 0.89%,85.34± 1.12%,82.82 ±0.87%,82.40 ± 0.93%and 80.40 ± 0.72%,respectively.The leakage rate was the lowest when stored in refrigerator at 4?(dark).The effect of artificial gastric juice on the stability of liposomes was greater than that of artificial intestinal juice.The results of DSC showed that HMA/liposome showed pre phase transition at the experimental concentration,and the addition of HMA at different mole percentages resulted in the increase of half peak width and phase separation of the main phase transition;with the increase of drug concentration,the main phase transition temperature and enthalpy of liposome fluctuated.SXRD results showed that the HMA/DPPC liposomes with different mole percentage concentration all formed lamellae liposomes,and all the liposomes were in the layered gel phase at 20 degrees.FTIR results showed that the addition of HMA had no effect on the characteristic peak position of DPPC.When 0<x?2.5 mol%,the peak area percentage of hydrated vC=O did not change;when 2.5<x?5 mol%,the peak area percentage of hydrated vc=o increased;when 5<x?10 mol%,the peak area percentage of hydrated vC=O decreased;when 10<x?25 mol%,the peak area percentage of hydrated vC=O did not change.5.Through the comparative analysis of entrapment efficiency and stability data of three kinds of drugs/DPPC liposomes,as well as the comprehensive comparison of DSC,SXRD and FTIR experiments overall,the influence on DPPC liposomes:apocynin is stronger than that of paeonol,and paeonol is stronger than that of HMA.Conclusion:The position of paeonol,apocynin and HMA in liposomes changed with the concentration,and they could act in the hydrophobic tail chain and hydrophilic head region of DPPC.Paeonol and apocynin can promote liposomes to form undulation gel phase at room temperature.HMA has no obvious effect on the phase of liposomes.Three kinds of drug molecules can increase the fluidity of lipid bilayer membrane. |
PDF Full Text Request