| 1Objective(1) Extracting theacrine from Camellia assamica var.kucha to meet the experimental needs;(2) Inspecting theacrine pharmacokinetic behavior characteristics in rat plasma;(3) Confirming the sleep-promoting effect of theacrine, and preliminarily explorating the relationship of the central nervous system role of theacrine and adenosine A1R, A2AR.2Methods(1) we extracted theacrine from Camellia assamica var.kucha by fixed, dried, crushed, solvent abstraction, chloroform extraction, column chromatography, preparative RP chromat-ographic separation and methanol crystallization.(2) First, determining pre-treatment method of the plasma sample, and optimizing the chromatographic conditions, and determining the analytical methods from the specificity, linearity, accuracy, precision and stability of the plasma samples.The Pharmacokinetics of theacrine in rat was investigated by the developed HPLC method. The main pharmacokinetic parameters were obtained. For the dose-dependent pharmacokinetic investigation different i.g. doses (30,10and3mg/kg) of theacrine were administered to three groups of rats.(3) We inspected the sodium pentobarbital-induced sleep time in mice after giving the mice different doses of theacrine and threshold dose of sodium pentobarbital to further prove the sleep-promoting role of theacrine.The adenosine A2AR antagonist (SCH58261) or the adenosine AIR antagonist (DPOX) was injected to the two groups of rats. Both of the two groups of rats were administered by medium doses i.g. of theacrine after10min, and compared the difference between each group.3Results(1) The purity of theacrine analyzed by HPLC is more than98%, which is complied with the requirements of the experiment.(2) We choose caffine as internal standard (IS). Extracted with ethyl acetate was employed for plasma sample pretreatment. The Chromatographic conditions were as follows: The separation was achieved on a Phenomenex Luna C18(4.6mm×250mm,5?m)coupled with a C1g guard column.The injection volume was20?l. The detection wavelength was290nm. The column temperature was maintained at25?. The mobile phase consisted of methanol:water=25:75at a flow rate of1.0ml/min.The assay was linear over the concentration range of0.5-100?g/ml with0.5?g/ml as the lower limit of quantification. It was sensitive, specific, accurate, precise and reproducible the extraction recoveries were (95.78±8.56)%,(92.55±5.02)%and (90.34±5.16)%when the concentration was0.5,5.0,50?g/mL. The assay was suitable for pharmacokinetic study of theacrine in rat.The results the pharmacokinetic study showed that plasma concentration of theacrine was high (Cmax35.45?g/ml after i.g.30mg/kg) and declined slowly with an elimination half-life (t1/2) from3to5hour. A good linearity (r=0.9841) was found in the regression analysis of the AUC0-t-dose. No significant difference of t1/2was observed in three groups.(3) The results showed that, the sleep time of10mg/kg and30mg/kg group were both extended, and the sleep time of30mg/kg group extended more notably. The sleep time of3.0mg/kg group was slightly lower than the blank group.The sleep time of the adenosine A2AR antagonist group were reduced on a certain extent compared with the control group. The difference of the adenosine AIR antagonist group and control group is not significant.4Conclusions(1) The kinetics of theacrine in rat was linear and was fitted to be one-compartment model after i.g. administration using Practical Pharmacokinetic Program-Version97(3P97).(2) Theacrine have the role of promoting sleep, and the mechanism may be by blocking the adenosine A2AR... |
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