Study On The Extraction, Conversion And Liposome Preparation Of Shikonin

The dry radix of Arnebiaeuchroma(Royle) Johnst and Arnebia guttata Bung belong to families of borage genus herb, it is mainly used for heat blood toxic, eczema, empyrosis in traditional Chinese medicine clinic. Modern pharmacological research demonstrate that Shikonin derivative show a reaction to antitumor activity such as lung cancer, liver cancer, gastric cancer, breast cancer, which is mainly run by Shikonin. But Shikonin derivative is restricted by low content in dry radix, almost insoluble in water and the poor absorption in body. The project of Shikonin has systematically studied on extraction, conversion and liposome preparation technology.Objective:1 Research for the extracting process of Shikonin derivatives2 Research for the best alkalized extraction conversion of Shikonin, and achieved high purity.3 Research for the preparation technology and quality evaluation of Shikonin liposome.Methods:1 The single factor experiment: With the quantity of Shikonin derivate as an index, to investigate extraction temperature, extraction time and solvent/drug(mL/g) for the determination of the main factors and level. Orthogonal test: With the quantity of Shikonin derivate as an index, it is used to confirm the best extracting process and process validation that carrying on three factors-levels orthogonal experiment and the analysis of variance statistics.2 The single factor investigation: With the Shikonin conversion quantity and its purity as an index, to investigate transforming temperature, transforming time, solvent/drug(mL/g) and the concentration of natrium hydroxydatum for the determination of the main factors and level. Orthogonal test: With the Shikonin conversion quantity and its purity as an index, it is used to confirm the conversion process and process validation that carrying on three factors-levels orthogonal experiment and the analysis of variance statistics. Structure validation: It is analysised by using HPLC technique to contrast the chromatogram of before and after, and combined with 1H-NMR, 13C-NMR, ESI-MS chromatogram data.3 Establish a HPLC method for the determination of Shikonin liposome. The single factor investigation: With Shikonin liposome encapsulation efficiency as an index, to investigate the solvent type, hydration medium type the drug/PhosphatidylCholine(mg/mg), and volume, water temperature, Phosphatidylcholine/Cholesterol(mg/mg), ultrasonic time for the determineation of main factors and level. Orthogonal test: With Shikonin encapsulation efficiency as an index, it is used to determine the best preparation process of Shikonin liposome and process validation that carrying on three factors-levels orthogonal experiment and the analysis of variance statistics. Shikonin liposome quality evaluation:using HPLC technique to measure Shikonin encapsulation efficiency and its release in vitro, TEM experiments with negative-staining for characterized appearance form, and measurement of the zeta value and particle size distribution is described by Malvern laser particle size analyzer.Results:1 The best extraction process:taking dry radix of Arnebia euchroma(Roylle) Johnst(smash into a diameter of less than 2cm) 40 g in a 500 mL conical flask joining 240 mL 95%Alcohol, heating extraction 4h at 50℃ constant water then vacuum filtering. The filter residue has extracted with 95%ethanol again at the same conditions. The 95%Athanol was removed under reduce pressure using a rotary evaporator. Finally the average quantity of Shikonin derivate was 2.20%.2 The best conversion: three accreted Shikonin derivate 100 mg dissolved in suitable amount of petroleum ether at 500 ml conical flask, then vacuum filtering. Sodium hydroxide 128 mL added into the filterate and heating 4h at 40℃ constant water, then vacuum filtering. The filtrate switched into a separatory funnel and added pure water to extracting not blue in the substratum. The alkaline-water layer processing by vacuum filtering was added formic acid for pH 5-6 and left for 12 h, then added petroleum ether extracting colorless in the alkaline water layer and evaporated the petroleum ether by reduce pressure using a rotary evaporator. Finally the average Shikonin conversion was 53.86% and 95.6% purity. It was shown that the conversion was Shikonin powered by structure identification date.3 The HPLC methodological study shows the method were reliable, can meet analytical requirement in vitro. The best preparation technology and prescription: Initially, the Phosphatidylcholine, Cholesterol together with Shikonin were dissolved in Petroleum Ether/methanol 3:5(v/v) at constant mass ratios 1:9 Shikonin / Phosphatidylcholine(mg/mg) and 7:1 Phosphatidylcholine/Cholesterol(mg/mg).The organic solvent was slowly removed under reduced pressure using a rotary evaporator, forming a thin lipid film in the inner side of the flask. The lipid film was then hydrated with 20 mL 0.9%Sodium Chloride for 1h.The system was filtered and ultrasonic for two 5min periods interrupted by a 5min resting period, the encapsulation efficiency of Shikonin liposome was 76.90% on average, the average particle size was 187.73 nm.The result of liposome quality evaluation described that the liposome of optimum preparation was round to oval, equable dispersiy, the size was relatively uniform and its about 200 nm, the zeta potential value was-38.94 mV, and sustained release characteristic.Conclusion:The quantity of Shikonin derivate was higher, and the processing was simple, which is worth to Shikonin conversion experiment for a preliminary material. The quantity and purity of Shikonin conversion has greatly increased than traditional method such as alkali acid or ethanol alkaline. The encapsulation efficiency of Shikonin liposome was higher, the quality indexes accorded with standard. The study has finished research systematically for the Shikonin, and would construct a foundation to the further development and utilization.