Study On Microemulsion Vehicle For Volatile Oil Of Danggui And Chuanxiong For Intravenous Administration And In Vivo Evaluation

Volatile oil is one of the common parts in Traditional Chinese Medicines with great contents and noticeable pharmacological effects, which is used extensively in some diseases such as cardio-cerebrovascular, tumor and virus aspects. However, because of poor solubility for most volatile oil, studies about its praeparatum through veins have great difficulties. In this study, the volatile oil extracted by steaming distillation from Danggui and Chuanxiong was chosen as model drug to develop an O/W microemulsion vehicle for intravenous administration, what is more, the physical and chemical properties, pharmacokinetics in rabbits and distribution in mice were investigated, too.Microemulsion has become one of the hot subjects of applied study for intravenous administration in pharmaceutics domains. Comparing to emulsion, liposome and micellar solution, drug delivery system of microemulsion offers considerable potential predominance as follows: it is a thermodynamically stable, isotropic transparent or semitransparent system, so it can pass through micropore filter, stand autoclave sterilization; the viscosity of microemulsion is rather low, which would lessen much pain during injection; the particle diameters of microemulsion are usually less than the that of red blood cells; the drugs entrapped in microemulsion will be released slowly, at the same time, it maybe has some targeting effects. However, the large amount of surfactant and cosurfactant in composition of microemulsion is the main barrier which blocks its more application in intravenous administration. Therefore, after selecting high effective and low toxic surfactant and cosurfactant, the forming condition of microemulsion can be optimized by pseudo-ternary phase diagrams to obtain a formula with less surfactant and cosurfactant and more volatile oil. At the same time, the microemulsion prepared should prevent phase transition when it is diluted by blood after injection.Z-ligustilide is the primary ingredient both in volatile oil of Danggui and Chuanxiong. But its extreme instability at room temperature restricts its application in pharmacy. In this study, before pharmaceutical experiments, the standard substance of Z-ligustilide was separated by gel silica column chromatography through elution twice, and its purity was more than 98% by HPLC. The structure was identified by IR, NMR, MS and UV methods to be Z-ligustilide.As mentioned before, Z-ligustilide is rather instable, and illumination is the major factor which induced its degradation. Other factors, which could affect its stability in aqueous solutions including pH value, composition of solvents, kinds and amounts of stabilizer, etc al, were detailedly investigated by accelerating experiments. The results indicated that the most suitable vehicle for Z-ligustilide included 20% 1, 2-propanediol and 0.30% stabilizer S, and its pH value was adjusted to 5.8. The degradation trend of Z-ligustilide was fitted according with Weibull probability distribution, and its shelf-life(T₉₀) was 1.77 years through Arrhenius equation by classical thermal methods, which was accordant to the facts.As the model drug in this study was volatile oil from Traditional Chinese Medicines, in which the lactone with carbon chain between 10～12 was more than 60%, it may serve as the oil phase of microemulsion per se, which could avoid the toxicity of other oils. Meanwhile, experimental results presented that, the addition of other oils such as soybean oil needs more surfactant and cosurfactant to form microemulsion, thus could increase the potential danger of preparations.Four surfactants including phospholipids, Poloxamer 188, Cremophor EL 35 and Solutol HS 15, which could be adopted by intravenous injection as reported were considered by pseudo-ternary phase diagrams, and results showed that Solutol HS 15 was more suitable to prepare microemulsion, furthermore, the microemulsion containing Solutol HS 15 could improve its stability and prolong existing time in blood circulation, which was more favorable for this preparation. The cosurfactant was ethanol and the mass weight ratio of surfactant to cosurfactant was fixed at 1:1, which could prevent appearance of liquid crystal and gels. The viscosity of microemulsion prepared is low enough for injection.According to all the information obtained form pseudo-ternary diagrams, the weight ratio of surfactant/cosurfactant to volatile oil was maintained as 7:3, thus, the O/W microemulsion domain could extended to the culminated point of water phase in diagrams, which suggested that the microemulsion would not occur phase inversion even was diluted by large amount of mediums. During study, the viscosity, electric conductivity, refractivity and particle diameters of samples containing different amount of water were reviewed respectively, and according to these results, the phase transition process was approached initially. When the amount of surfactant and cosurfactant was fixed, the composition with maximum volatile oil was determined. Moreover, the particle diameters would not enlarge when it was diluted. The surrounding temperatures would affect the shape and size of O/W microemulsion in diagrams to a certain degree, however, there existed some common domains under different temperatures, which could be predicated to be less influenced by temperature, and this could simplify the preparation process. The O/W microemulsion domain had no change when the distilled was substituted by normal saline, which proved ionic addictives would not affect the formation of microemulsions. The photo under transmission electron microscope displayed the microemulsion drops as spherical particles with average diameter of 110nm, and dilution by normal saline or water would reduce its diameters.The content of Z-ligustilide in microemulsion was determined by HPLC. The stability of microemulsion was investigated by viscosity, electric conductivity, refractivity, particle diameters and content of Z-ligustilide and results of accelerating testing（40℃） and room temperature showed it was basically stable during 6 months.Through single intravenous injection to mice, the median lethal dosage（LD50） of microemulsion was 321.04mg/kg, and the 95% confidence interval was 239.44～430.45mg/kg treated by Bliss procedure.There were few reports about the in vivo study about Z-ligustilide in volatile oil of Danggui and Chuanxiong. A protein precipitation method using 2 times of acetonitrile was developed to treat the samples of plasma and biological tissues, and the pharmacokinetics of Z-ligustilide in rabbits and tissue distribution in mice were investigated by intravenous injection. The distribution of Z-ligustilide in microemulsion was fitted a two-compartment model in rabbits, and t_1/2α and t_1/2β was 1.37min and 10.48min, respectively, and the area under curves（AUC） was 28051.56 ng min/ml. However, when the volatile was administrated as 1, 2-propanediol solution, no drug could be detected after 4min. The results of tissue distribution in mice showed that AUC of microemulsion in plasma was 12 times more than that of solution, and the distribution of Z-ligustilide in every tissue was uniform. However, the concentration of Z-ligustilide in plasma was only sixth to that of microemulsion soon after the volatile oil solution was injected; other drugs were found mostly enriched in kidney. The distribution of Z-ligustilide in mice plasma was fitted a two-compartment model, which was accordant to that in rabbits.As the basis of pharmacokinetics of Z-ligustilide both in rabbits and mice after the volatile oil microemulsion was administrated by intravenous injection, it was found that, although the existence of Z-ligustilide in plasma had been greatly improved, it would be eliminated from plasma quickly. This fact meant that it was difficult for Z-ligustilide to remain an effective concentration. Thus, the volatile oil microemulsion was recommended to be administrated by intravenous infusion after it was diluted to certain concentration, and this method might profit the pharmacodynamic action and reduce its stimulation.