| Aim To develop a freeze-dried tanshinone ? A micro-emulsion system using Kolliphor HS15 and Polysorbate 80 as solubilizers.In order to obtain a excellent water-soluble and stable tanshinone ?A injection,the freeze-drying process,physical and chemical characterization and electrochemical behavior were investigated.Methods1.Constraction of tanshinone ?A micro-emulsionsThe determination method of tanshinone ? A content was established and the dissolution of tanshinone ? A encapsulated in micro-emulsion was selected as the preparation screening index;By observing the critical point that the solution changed from turbidity to clarity or clarity to turbidity,the pseudo-ternary phase diagram was used to screen the types and proportions of surfactants,and to reduce the dosage of Polysorbate 80 and the side effects of the product;The preparation was optimized by design-response surface method and determining the proportion of oil phase and surfactant to obtain the best preparation.2.Investigation on the freeze-drying process of tanshinone ?A micro-emulsionsThe optimum freeze-drying process was determined by the lyoprotectant,eutectic point,glass transition temperature and collapse temperature.The effects of freeze-drying on the stability of tanshinone ? A micro-emulsion system were investigated by comparing the freeze-dried sample,after freeze-dried sample and annealed sample in the aspects of surface tension,cloud point,particle,light transmittance,compatibility stability and chemical stability.3.Characterization of tanshinone ?A lyophilized micro-emulsionsThe physicochemical properties such as appearance,re-solubility,conductivity,particle size and zeta potential of tanshinone ? A micro-emulsion were characterized by using dynamic light scattering technique,differential scanning calorimetry and X-ray diffraction etc.4.Electrochemical behavior of tanshinone ?A lyophilized micro-emulsionsThe electrochemical determination of tanshinone ?A micro-emulsions was established by selecting the working electrode,supporting electrolytes,pH,scanning rate and so on.Furthermore,the redox mechanism on the surface of glassy carbon electrode was investigated by the effect of scanning rate.Result1.We used 269 nm as the determination wavelength,and the calibration curve between concentration and absorbance was as follow: A = 0.1154 C + 0.0040(R2 =0.9995),showing a good linear relationship for its concentration from 1.00 ?g/mL to8.00 ?g/m L.The feasibility of method was verified by recovery test,precision test,repeatability test.The optimum conditions were obtained as follows: 22.11% GTCC,HS15: PS80(Km = 2.18),and the solubility of tanshinone ?A was 86.4 times higher than that of API.2.The amount of lyoprotectant was 15%,the eutectic point of the micro-emulsion was about-9 ?,the glass transition temperature of freeze-dried sample and annealed sample were-38.25 ? and-38.52 ?,and its collapse temperature were-28.8 ? and-33.1 ?,respectively.The final freeze-drying process is: pre-freezing temperature was settled at-40 ?,keeping pre-freezing for 4 h;when cold trap temperature dropped to-55 ?,heating shelf temperature to-25 ? for 1 h;and then the temperature was raised by 10 ? every time until the sample temperature reaches to 25 ? and kept for 6 h to obtain the freeze-dried sample.The preparation process is simple and easy to operate.By comparing lyophilized,annealed and not-lyophilized sample in terms of surface tension,cloud point thermodynamics,insoluble particles,light transmittance,compatibility stability and chemical stability,and the result suggested that the stability of lyophilized and annealed samples were superior to not-lyophilized samples.3.Tanshinone ? A micro-emulsion prepared by the optimal conditions had excellent appearance,and presented a orange-red transparent liquid after reconstituting with water,5% dextrose solution,10% dextrose solution or 0.9% sodium chloride solution,whose mean particle size were(21.66 ± 0.23)nm,(24.42 ± 0.42),(27.06 ±0.26)nm and(21.92 ± 0.18)nm,respectively,and the polydispersity index were(0.166 ± 0.12)nm,(0.064 ± 0.08),(0.092 ± 0.11)and(0.062 ± 0.03),respectively;The micro-emulsion was O/W and the pH value was between 6 and 7;DSC and XRD results showed that tanshinone ? A was encapsulated in the micro-emulsion structure in molecular form.4.Tanshinone ?A micro-emulsion had a quasi-reversible redox reaction on glassy carbon electrode.The optimum reaction conditions were as follows: 5 mol/L pH 4.0acetate buffer was as the support electrolyte,the scanning potential range was 0.4~-0.6V,scanning rate of 50 mV/s,the number of electron transfer was 2,the electrode reaction was jointly controlled by diffusion and adsorption.The concentration of tanshinone ?A and the reduction peak current showed a good linear relationship in the range of 0.2-1.2 ?g/mL: Ipc(?A)= 0.6420 C(?g/mL)+ 0.2612,R2 = 0.9977.The limit of detection and the limit of quantification were 50.25 ng/mL and 167.5 ng/mL,respectively.The recoveries,precision and accuracy of the method were proved to be feasible.ConclusionThrough the selection and optimization to the composition and proportion of tanshinone ? A solubilization system,the tanshinone ? A micro-emulsion system showing excellent water-solubility was constructed.The stability of the preparation was improved by the combination of the freeze-drying technology,and a novel tanshinone? A lyophilized micro-emulsion system was developed.At the same time,the electrochemical redox mechanism was studied and the electrochemical determination method of tanshinone ? A was established,which all provided a new idea for the detection of electroactive substances. |
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