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Objective To develop a HPLC-MS/MS method for the determination of aconitine and study thein vitro metabolic stability of aconitine in dog tissue homogenates.Methods The chromatographic separation was performed on a C18 column.The mobile phase consisted of acetonitrile and water with 0.2％ formic acid and 5 mmol/L ammonium acetate.A triple quadrupole tandem mass spectrometer equipped with an electrospray ionization interface source was used for the quantitative determination in the positive selective reaction monitor mode.Aconitine was incubated with dog tissue homogenates and samples were withdrawn at different time points and precipitated by acetonitrile with internal standards citalopram.Results Aconitine showed good linear relationship over the range from 5 to 500 ng/ml.The recoveries of aconitine were between 85.73％ and 92.12％ at three QC concentration levels.The intra- and inter-day precisions were 5.32％ - 8.95％ and 5.45％ - 8.86％,respectively.After incubation,about 20％ of aconitine were cleared in the liver and small intestine,and t1/2 were 460.6 and 521.3 min,respectively.But none was metabolized in the stomach and kidney.Conclusion These results demonstrated that aconitine was mainly metabolized in the liver and small intestine at a slow rate.
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OBJECTIVE To explore the absorption mechanism of thiophenorphine, and its effect on P-glycoprotein (P-gp) expression by using Caco-2 cell monolayer model. METHODSThe LC-MS-MS method was applied to determine thiophenorphine concentration in millicell system. The bi-directional permeability studies were performed to investigate the potential involvement of efflux carriers in the intestinal absorption. P-gp inhibition was studied by flow cytometry using calcein-AM as P-gp substrate.The expression of P-gp was evaluated using Western blotting. RESULTSThiophenorphine transport in Caco-2 cells was in time-dependent manner. Its average apparent permeability coefficient (P_(app)) was 2.338×10~(-6) cm·s~(-1). P_(app) was increased 2.8 folds by P-gp inhibitor ciclosporin A, and 2.3 folds by mulitdrug resistance-associated protein2 (MRP2) inhibitor MK571. The accumulation of calcein-AM and the expression of P-gp in Caco-2 cell line wasn't changed noticeably by thiophenorphine. CONCLUSION Thiophenorphine is a common substrate of P-gp and MRP2 and it shows normal transport in millicell system. The expression of P-gp doesn't induce by thiophenorphine.
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Objective To investigate the effects of anticholinergic antidote and rhodosin on the brain injury induced by soman intoxication combined with hypobaric hypoxia in rats. Methods A total of 72 Wistar rats were divided into 4 groups: hypoxia control (HC), hypoxia plus soman (HS), hypoxia plus soman plus anticholinergic antidote (HSAA), and hypoxia plus soman plus anticholinergic antidote plus rhodosin (HSAAR). The animals after soman intoxication (72 ?g/kg) were placed in a hypobaric (62 kPa) apparatus for hypoxic exposure for 48 h. Rats were sacrificed for brain tissue detachment at the time points of 12, 24, and 48 h. Evans blue (EB) content and PLA 2 activity were detected biochemically. CaM concentration was determined by radioimmuno assay. Results Compared with the rats in HC, soman induced significant increases of brain EB, PLA 2, and CaM at 12, 24, and 48 h in HS. Elevated EB, PLA 2, and CaM induced by hypoxia and soman intoxication in rats in group HSAA were obviously attenuated by anticholinergic antidote. More significant decreases of brain EB, PLA 2, and CaM were found in rats in group HSAA. Conclusion Both anticholinergic antidote and anticholinergic antidote plus rhodosin have the preventive effect on rat brain damage induced by soman intoxication combined with hypoxia.
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Objective To investigate the effect of soman on the Janus kinases (JAKs) expression in cell line PC 12 . Methods The PC 12 cell was used in these experiments and treated with soman at a concentration of 20 ?mol/L . RT PCR and Western blotting were employed to detect the mRNA and protein expressions of JAK1, JAK2 and JAK3 at the time points of 0, 6, 12 and 24 h. The products were sequenced by Sanger's double strand DNA sequence determination. Results The expression levels of JAK1, JAK2 and JAK3 mRNAs and proteins increased at 2 h, reached the highest at 12 h and decreased at 24 h, but they were still higher than those of the control. It was shown that the sequences of amplification products by RT PCR were the same to corresponding ones in GenBank. Conclusion Soman intoxication enhances the expression of Janus kinases in PC 12 cells. JAKs genes may play an important role in brain injury due to soman intoxication.
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Objective To illustrate the features of soman induced signal transducers and activators of transcription (STATs) gene and protein expressions in cell line PC 12 . Methods The expression levels of STAT1, 3 and 5 mRNAs and protein in PC 12 cells were detected by semi quantitative RT PCR and Western blotting. PC 12 cells at 5～8 passages were randomly divided into 5 groups: control, intoxication groups for 2, 6, 12 and 24 h respectively. The products were sequenced by Sanger's double strand DNA sequence determination. Results The expression levels of STAT1, 3 and 5 mRNAs and proteins increased in PC 12 cell at 2 h and reached the highest at 12 h, then decreased at 24 h, but they were still higher than those of the control. The sequences of amplification products by RT PCR were the same to those in GenBank. Conclusion Soman intoxication can enhance the expression of STATs in PC 12 cells. STAT genes may possibly play an important role in brain injury.
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Object To study the pharmacokinetics of loganin in LIUWEI DIHUANG DECOCTION (LWDHD) in mice. Methods HPLC-UV detection was used to determine the loganin levels in biological samples. Results After ig LWDHD in mice, the plasma concentration-time course fitted well to one-compartment model with the 1st order absorption and with the following pharmacokinetic parameters; Ka= 0.04min -1, Ke=0.019 min -1, T (peak)=42.4 min, t 1/2ka=17.4 min, t 1/2ke=35.75 min. Conclusion The pharmacokinetic parameters of loganin obtained in the study may be attributed to the effect of other constituentsin the DECOCTION.
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The fifth muscarinic receptor (M5), the last one of the mus ca rinic receptor family to be cloned, has the same basic formation characterizatio n as G-protein coupled receptor family. M5 transduces signals by coupling with G-proteins, which then modulate the activities of a number of effector enzymes and ion channels. As M5 also plays a variety of prominent physiological roles by regulating central transmitters NO and DA, it has been considered as a novel dr ug therapy target for drug addiction, dysfunction of dopamine-ergic nervous sys tem, Alzheimers disease and cerebral ischemia.