Studies On Compound Brucea Javanica Oil Submicron Emulsion Injection

Brucea javanica oil （BJO） and Coix seed oil （CSO） are the fixed oil obtained from the fruits of Brucea javanica （L.） Merr. and the seeds of Coix lacryma-jobi L. var. mayuen （Roman.） Stapf., respectively. In this paper, compound Brucea javanica oil submicron emulsion injection （BCOE） was prepared and evaluated in order to develop a compound traditional Chinese medicine for anti-cancer application, which could alleviate toxicity and raise their efficacy when combined with radiotherapy or chemotherapy and used before surgical operation.After refined, BJO and CSO were studied on their physical and chemical properties and they were consistent with the requirement for injection. An HPLC method combined with precolumn derivatization was developed for the assay of linoleic acid, palmitic acid, oleic acid and stearic acid in BJO and CSO. Their contents were 17.35%, 8.65%, 62.24%, 5.51%; and 21.18%, 11.85 %, 50.85%, 2.93%, respectively. The influencing factor test showed that BJO and CSO were stable at 40℃and unstable exposed to light. The optimal ratio for intravenous administration was set down as 3:1 on the basis of the effect of BJO and CSO with different ratios on S₁₈₀ sarcoma-bearing mice.Based on the typical formulation of fat emulsion, high pressure homogenization was developed to prepare BCOE using lecithin and sodium oleate as emulsifier and stabilizer, respectively. The single factor test was used to investigate the influence of process parameters （including temperature, pressure and cycles of high pressure homogenization, and sterilization condition） and formula composition （including the amount of lecithin and cosurfactant such as tween 80, F-68 and sodium, and pH） on the resulting emulsion properties such as particle size and distribution,ζ-potential, stability parameter K_E, and pH. The basic formulation and preparation technology of BCOE was: oil phase was composed of BCO （7.5%） and CSO （2.5%）, the amount of lecithin, tween 80 and sodium oleate were 1.2%, 0.1%, and 0.03%, respectively. High homogenization condition was 800bar, 6 cycles, and pH was adjusted to 8.0 before sterilization at 115℃for 30 min.BCOE was prepared base on the optimal formulation and preparation technology. BCOE was studied on characteristics, identification, particle size and distribution, pH,ζ-potential, and fatty acids contents. The mean particle size, pH andζ-potential were 213.7±50.0 nm, 7.51 and -30.24 mV, respectively. The contents of linoleic acid, palmitic acid, oleic acid and stearic acid were 13.07, 8.98, 44.04, and 3.30 mg/g, respectively. The above results provided the foundation for establishment of quality control system of BCOE.Shaking test and freeze-thaw cycling test revealed that the stability of BCOE would be influenced by severe shaking and temperature change and should be avoided severely shaking and changing of temperature and be prohibited freezing during storage and transport. The long-term stability study showed that CSOE was stable under 40℃for 3 months, 25℃for 6 months and 6℃for 18 months.An UPLC-MS method was developed and validated for the simultaneous determination of triolein and oleic acid in rat plasma and applied to the pharmacokinetics of BJOE. BCOE （10, 7.5, 2.5 ml/kg）, BJOE （7.5 ml/kg） and CSOE （2.5 ml/kg） were administered to rats via femoral vein. The pharmacokinetic parameters of triolein and oleic acid were evaluated by the non-compartmental method. Based on the AUC, the pharmacokinetics process of oleic acid was in accord with linear pharmacokinetics, however, triolein showed non-linear pharmacokinetics characteristics in 2.5-10 ml/kg dose ranges. The pharmacokinetics of triolein for BCOE high dose group were most adequately described by Michaelis - Menten equation. The average values for V_max, K_M, and volume of distribution （V） were 31.99±7.95μmol/L/min, 878.32±345.96μmol/L and 60.55±8.29 ml/kg, respectively. Triolein in other groups with 2.5-10 ml/kg dose ranges demonstrated linear pharmacokinetics. The AUC and C_max of triolein and oleic acid increased with the increasing of dose in BCOE groups and those of high dose group were significantly higher than for low dose group （P<0.05 or P<0.01）. The Cl of triolein and oleic acid for high dose BCOE group were significantly smaller than those of medium and low dose BCOE group （P<0.05 or P<0.01）. There were marked differences in AUC, V, Cl of triolein and t_max of oleic acid between high dose BCOE and BJOE groups （P<0.05 or P<0.01）. Significant differences in AUC, C_max, t_max, V and Cl of triolein and AUC, t_max and V of oleic acid were observed between high dose BCOE and CSOE groups （P<0.05 or P<.01）. Significant differences in pharmacokinetics parameters were mainly attributed to the changes of metabolic pathway of triacylglycerols in rats with different dose administered. The establishment and application of this method in this paper were of innovative significance internationally. There was few corresponding reference internationally.The safety experiment results of BCOE indicated that the in vitro haemolysis test, the blood vessel irritation test, active cutaneous anaphylaxis and passive cutaneous anaphylaxis test were all negtive. Acute toxicity result showed that mice were all survive and had no significant toxic symptom after intravenous administered the maximal tolerance dosage, which was 240 times as much as the clinic dose of man. The results demonstrated that BCOE would be safe in clinical treatment.After administered 10 ml/kg for 7 days, the tumor growth inhibitory ratio （TGIR） of BCOE on S₁₈₀ sarcoma-bearing mice were 44.2%-49.8% and higher than those of BJOE and CSOE. BCOE could prolong the survival period of EAC-bearing mice and percentage increased life span （% ILS） was 29.4%-38.7%, which was lower than that of CSOE but higher than that of BJOE. The antitumor activity of BCOE was dose-dependent and obviously higher than oral application. BCOE could alleviate toxicity of CTX and 5-Fu on normal mice and increase anti -tumor efficacy of CTX on S₁₈₀ sarcoma-bearing mice. The antitumor activity study demonstrated that BCOE had antitumor activity and could alleviate toxicity and increase efficacy when combined with chemotherapy. Few corresponding reference was found internationally.