Glucose-modified Compound Liposomes With Cyclovirobuxine D Ligustrazine Research On Its Optimized Preparation Process

ObjectiveCyclovirobuxine D,also known as Huangyangning,Cyclovirobuxine D,xanthine and so on,is an alkaloid extracted from Euonymus microphylla and its allied plants.It has pharmacological effects such as neuron protection,anti-arrhythmia and myocardial ischemia.It is often used in the treatment of coronary heart disease,angina pectoris and cardiac insufficiency.ligustrazine is an active component extracted from the rhizome of Ligusticum Chuanxiong,an umbrella plant of Chinese medicine.It belongs to amide alkaloids.It has the pharmacological effects of expanding peripheral blood vessels,lowering blood pressure,improving cerebral circulation,inhibiting platelet aggregation and anti-thrombosis.It is widely used in the treatment of ischemic heart and cerebrovascular diseases.The aim of this study is to enhance the bioavailability of Cyclovirobuxine D and Ligustrazine Hydrochloride by using a new type of targeting agent,liposome,and enhance the brain targeting advantage of liposome through glucose modification.Methods1.Establishment of an analytical method for Cyclovirobuxine by D-HPLC-MS/MSIn this study,an analytical method of Cyclovirobuxine D-HPLC-MS/MS was established,and the specificity,standard curve,precision and stability of the chromatographic conditions were investigated.2.Establishment of HPLC analysis method of Cyclovirobuxine D pre column derivatization.The HPLC method of Cyclovirobuxine d-precolumn derivatization was established,and the specificity,standard curve,precision and stability of the chromatographic conditions were studied.3.Establishment of an analytical method for Ligustrazine Hydrochloride by HPLC-UVThe chemical structure of Ligustrazine is tetramethylpyrazine.In this study,a method for the determination of Ligustrazine Hydrochloride by HPLC was used.The specificity,standard curve,precision and stability of the chromatographic conditions were investigated,4.Preparation and Pharmaceutical Evaluation of Compound Liposome with Cyclovirobuxine D ligustrazineLiposomes were prepared by film dispersion-pH gradient method.The encapsulation efficiency was taken as an index.The mass ratio of lecithin to cholesterol,the mass ratio of lecithin to drug,the time of film formation,the hydration temperature and time,the type of hydration solution and the pH gradient were investigated by single factor.The morphology,particle size,potential and stability of the liposomes were also studied.5.Preparation and Pharmaceutical Evaluation of Compound Liposome Modified by GlucoseThe structure of cholesterol-sebacic acid-glucose ester(CHS-SE-GLU)was confirmed by mass spectrometry,and the success of enzymatic synthesis of glucose ligands was determined.Orthogonal design was used to optimize the formulation ratio of Liposome Targeted by Colpound with Cyclovirobuxine D ligustrazine.The morphology,particle size,potential and stability of the liposomes were also studied.Results1.The analytical method of Cyclovirobuxine D by HPLC-MS/MS was established.Chromatographic conditions:chromatographic column:Waters Xterra MS C18(2.1*50 mm 3.5μm);mobile phase:acetonitrile(A)-0.01mol/L ammonium formate solution containing 0.1%formic acid(B),gradient elution;flow rate:0.35 mL/min;column temperature:30℃;injection volume:5μL.Mass spectrometry conditions:ion source:electrospray ionization source(ESI);ion mode:positive ion mode;capillary voltage:4.0 kV;nozzle voltage:500 V;dryer temperature:300℃;drier flow rate 5 L/min;sheath gas temperature:250℃;sheath gas flow rate:11 L/min;scanning mode:multiple reaction monitoring mode(MRM),detection ion pair m/z403.4→372.1,(quantitative ion pair);Fragmentor:82 V;collision energy:25 eV.The method has high specificity,high precision and good repeatability,which meets the requirements of quantitative analysis of Cyclovirobuxine D.2.HPLC method for the determination of Cyclovirobuxine D was successfully established.Chromatographic conditions:instrument:Shimadzu LC-20A;chromatographic column:Agilent C18(4.6*250mm 5μm);mobile phase:methanol:water=83:17;flow rate:1ml/min;detection wavelength:240 nm;column temperature:30℃;injection volume:20 μL.The method has the advantages of high specificity,high precision and good repeatability,and meets the requirements of Cyclovirobuxine D quantitative analysis.3.A method for the determination of Ligustrazine Hydrochloride by HPLC was established.Chromatographic conditions:instrument:Shimadzu LC-20A;chromatographic column:Agilent C18(4.6*250 mm 5 μm);mobile phase:methanol-water=65:35;flow rate:0.7 mL/min;detection wavelength:280 nm;column temperature:30℃;injection volume:20 μL.The method has high specificity,high precision and good repeatability,which meets the requirements of quantitative analysis of Ligustrazine hydrochloride.4.Optimizing the preparation process of compound Liposome with Cyclovirobuxine D ligustrazineAfter investigation,it was found that when lecithin:cholesterol=7:1;lecithin:vitamin E=20:1;lecithin:octadecylaminc=20:1;lecithin:Cyclovirobuxine D:ligustrazine hydrochloride=100:10:2.5,it was dissolved in anhydrous ethanol by ultrasound,evaporated under pressure at 45℃ for lh,and then hydrated to 20 mg/ml of lecithin by pH=3 disodium hydrogen phosphate citric acid buffer solution;probe ultrasound 3 s stopped 3 s for 3 minutes;after ultrasound,liposome passed 0.22 μm microporous membrane three times,solution adjusted to pH=9 by 1 mol/L NaOH,incubated at 60℃for 5 min,the highest encapsulation efficiency of liposome was obtained,the encapsulation efficiency was ligustrazine hydrochloride(80.15±2.31)%,Cyclovirobuxine D(99.95±0.03)%.At the same time,the quality of compound Xionghuang liposome was evaluated.The results showed that its particle size was(117.97±0.93)nm,zeta potential was(2.59±0.28)MV,PDI was(0.226±0.01).4.5.Successful preparation of glucose-based ligandsGlucose-based ligand cholesterol-sebacic acid-glucose ester was synthesized by enzymatic reaction using vinyl acetate,sebacic acid,cholesterol and glucose as raw materials.ESI-MS m/z 755.59[M+Na]+was shown by mass spectrometry,which was consistent with the literature description.6.Optimized Liposomes Targeted by Compound with Cyclovirobuxine D ligustrazineOn the basis of the preparation technology of compound Ligustrazine ordinary liposomes,the formulation was optimized by orthogonal design:the mass ratio of lecithin to cholesterol was 8:1,the mass ratio of lecithin to ligustrazine hydrochloride to cyclovirobuxin D was 100:10:2.5,and the dosage of cholesterol-glucoside was 2%.The liposomes with average particle size of(89.95±0.44)nm,encapsulation efficiency of tetramethylpyrazine hydrochloride(64.91±1.80)%and encapsulation efficiency of Cyclovirobuxine D(99.93±0.05)%were prepared.ConclusionIn this study,the preparation process of compound with Cyclovirobuxine D ligustrazine ordinary liposomes was successfully optimized by single factor investigation,so as to improve the entrapment efficiency of drugs in the liposomes.Through orthogonal design experiments,a reasonable and stable preparation process of compound with Cyclovirobuxine D ligustrazine targeting liposomes was obtained.Provide preparation basis for follow-up research.