| Objective:Through establishment of ion-pair RP-HPLC method in arecoline detection,in order to provide an experimental means for a comprehensive assessment of the toxicity and mechanism, dose-response relationship,as well as pharmacokinetics studies etc of arecoline.Methods:Using Japanese Shimadzu HPLC(Lcsolutin workstation),0.1%SDS(containing 0.1%triethylamine,phosphoric acid adjustment pH to 2.5～2.6) and acetonitrile as mobile phase, arecoline content of liver homogenate and Arecoline nut,as well as in vitro metabolism rate of arecolin was detected.Arecoline cytotoxicity was evaluated by MTT method and morphological observation.Effects of microsomal enzyme and FMO in metabolism of arecoline and cytotoxicity induced by arecoline was explored through the 50℃heat treatment liver S9 or without NADPH in reaction system.Results:The minimum detection limit was 1ng,theory plate number was 15961±276,the injection magnitude and peak area showed good linearity in the 1～40ng dose rang with the ion-pair RP-HPLC method in arecoline detection.It was a good treatment process for extraction arecoline in liver homogenate,with 10%perchloric acid extraction,then ether extraction after ammonia basification,the minimum detection limit was 50ng/ml in liver homogenate.Arecoline in arecoline nut can be effective extracted with 2%hydrochloric acid,extraction rate was more than 99%after repeat 2 times of extraction,and it was easy and convenient for acid extract can be directly used to detect.In the vitro metabolic system with liver S9 or with the 50℃heat treatment of liver S9,regardless of with or without NADPH,Arecoline was rapidly metabolized.Therer was no significant differences in metabolism ability in different reaction system.half metabolism time was about 60～70min. Arecoline 50μg/ml had an obvious cell toxic effects,including growth and proliferation and MTT metabolism ability of L-02 liver cell,and an obvious dose-dependent manner can be observed in arecoline-induced cell injury.Compare with alone arecoline treatment group,the group with liver S9 pretreatment whether from the cell morphology,or from the cell MTT metabolism ability,showed protection cells from the arecoline-induced toxicity in a certain extent.By the liver S9 treatment, L-02 cells can tolerance 300μg/ml toxic dose of arecoline,after 50℃heat treatment for one minute,liver S9 still have the same protective effect,there was no significant difference in comparison with untreated liver S9.Conclusion:1.Ion-pair RP-HPLC was a high sensitivity and good reproducibility in determination of arecoline,and can be used as an better ideal approach for trace arecoline detection.By changing the composition of eluents,it can be used in the determination of arecoline in liver homogenate and betel nut.2.Liver homogenate fraction has strong ability of arecoline metabolism,metabolic rate was changed with time exponentially increase,the fitted non-linear regression equation was y=98.97e-0.0110X. Microsomal enzyme,FMO had no obvious effect in the arecoline metabolism.3.Arecoline was toxic to cells,in the dose of 50μg/ml,arecoline induced obvious toxic effects on the L-02 liver cells,and a clear dose-dependent can be observed.4.Liver S9 can protect L-02 liver cells from the toxicity of arecoline,0.05mg/ml of liver S9 almost completely blocked the toxic effects of 300μg/m arecoline.But the microsomal enzyme,FMO had no obvious effect in the process of metabolism detoxification of arecoline.
|
PDF Full Text Request