| Amlodipine?AML?,third-generation dihydropyridine Ca2+ channel blocker,inhibits Ca2+ entry into myocardial and vascular smooth cells by blocking the “L-type potential action” calcium channel,which leads to relaxed smooth muscle,decreased myocardial contractility and frequency,and slowed conduction.In addition,AML can lower the blood pressure by improving the activity of calcium pump in the cell membrane and reducing brain free calcium concentration.AML is completely absorbed,which is higher than other calcium antagonists but takes longer time.The peak plasma concentration is at 612 h of oral administration.Therefore,AML is characterized by long duration in lowering blood pressure and small peak fluctuation of blood pressure,which delays the occurrence of hypertension and reduces the extent of hypertension.Due to its medicinal value,Ginkgo leaf has been widely used in clinical practice for the prevention and treatment of various cardiovascular and cerebrovascular diseases including hypertension,coronary heart disease and angina.It is commonly used in combination with other cardiovascular drugs in clinical setting.Ginkgo leaf tablets combined with AML has been shown to significantly reduce sDR4 and s DR5 levels in the plasma and improve the myocardial cell apoptosis in elderly patients with hypertension.However,to the best our knowledge,the effect of ginkgo leaf on AML has not been reported from the perspective of drug metabolism.It is of great significance to investigate whether ginkgo leaf tablets have an impact on its pharmacokinetics.With the development of society,drinking has become a kind of living habits of human beings.White spirit,beer,red wine and champagne affect both lives and health of people.Ethanol is absorbed into the blood through the stomach,and its concentration is higher in the gastric smooth muscle than in the blood,thus affecting gastrointestinal tract.Studies have shown that ethanol can directly damage the gastric muscle,and produce toxic effects on the smooth muscle of the gastrointestinal tract.In the present study,we studied gastric funds,gastric body,cardia and pyloric circular muscle with experimental methods in pharmacology.In addition,pharmacological characteristics of the muscarinic?M?receptors and 5-hydroxytryptamine?5-HT?receptors-mediated contraction in gastric smooth muscle was also explored.However,the effect of ethanol on isolated gastric fundus,gastric body and circular muscle of the cardia and the pylorus of rat stomach has been incompletely understood.Part one: The effect of Compound Chinese Medicine ginkgo leaf tablets on pharmacokinetics of AML in ratsObjectives: To develop a UPLC-MS/MS method for the determination of AML and its metabolites based on HPLC-Orbitrap-Fusion method in measurement of metabolites of sulfonic acid amlodipine,and to explore the effect of ginkgo leaf tablets on pharmacokinetics of AML by comparing the pharmacokinetic parameters of AML and its metabolites in rats administered intragastrically with AML alone or AML in combination with ginkgo leaf tablets.Methods: Chromatography conditions: The samples were separated on a chromatographic column of Xbridge C18?100 mm×3 mm,3.5 ?m?.The column temperature and sample room temperature were maintained at 30 °C and 4 °C respectively.A mobile phase consisting of acetonitrile?A?and water with 0.1% formic acid?B?was used at a flow rate of 0.6mL/min.The injected volume was 2 ?L.The gradient elute was as follows: 0min,30% B;3.5min,50% B;8.5min,80% B;10min,90% B;10.5min,90% B;11min,30% B;Prazosin was used as an internal standard.Mass spectrometry conditions: UPLC-MS/MS was used.The mass spectrometer was operated in the positive electrospray ionization mode with an electrospray ionization interface?ESI?;Quantification was performed using multiple reaction monitoring?MRM?.The capillary voltage was 3 kV;and the ion source temperature was set at 150 °C.The appropriate analytes and parameters of internal standard were use to determine the parameters,such as ion pair,dwell time,scanning time,cone voltage and collision voltage.HPLC-Orbitrap-Fusion: The mass spectrometer was operated in the positive electrospray ionization mode with ionspray voltage 3.5 kV,and negative electrospray ionization mode with ionspray voltage2.5 kV.The other parameters were as follows: sheath gas: 40 Arb;aux gas: 12 Arb;sweep gas: 1 Arb;Ion Transfer Tube Temp: 330 °C;Vaporizer Temp: 315 °C;scan range: 100-600 m/z;mass resolution: 60000.Experiment: The types and structures of metabolites were analyzed using HPLC-Orbitrap-Fusion and Mass Frontier 7.0 software.Twenty rats were randomly divided into two groups: control group?n=10?and experimental group?ginkgo leaf tablet group;n=10?.Rats in control group and ginkgo leaf tablet group were given intragastrically with distilled water and ginkgo leaf tablets of 2 mL respectively every day for 15 consecutive days.On day 16,AML was administered intragastrically at a dose of 1 mg/Kg?2 mL?to all rats.Rats were fasted for 12 h and allowed free access to water before administration.Blood samples?0.5 mL?were collected from plexus venous at fundus oculi at 0,0.5,1,1.5,2,3,4,6,8,12 and 24 h after administration as well as 0.5 h prior to administration,followed by storage in heparin anticoagulant tube.The plasma samples were centrifugated and stored at-20 °C for future analysis.The blood concentration of AML at the eyes in each rat and the peak area of metabolites were determined.Data analysis and statistical analysisAll pharmacokinetic parameters were analyzed using WinNonlin software.All data were expressed as mean±SD.Two-factor variance statistics analysis?ANOVA?was performed.The main pharmacokinetic parameters between the experimental group and control group were analyzed using statistical analysis software Graph Pad Prism 5.0 and Graphpad Instart 3.0.P value less than 0.05 was considered to be statistically significant.Results: The retention time of AML and prazion was 3.48 min and 1.13 min respectively.There was no endogenous interference from plasma in the peak and determination of AML and internal standard substance.The calibration curve was Y=0.7887C+0.3851?R2=0.9996?,and the linear range and the LLOQ were 0.15-60 ng/mL and 0.15 ng/mL respectively.The RSD values of intra-day and inter-day concentrations for AML?0.3 ng/ mL,5 ng/ mL and 30 ng/ mL?were all less than 15% and the average recoveries of the three were 67.67%,70.00% and 80.16%,respectively.The RSD of average matrix effectsvwas 109%,100% and 100%,respectively.Plasma samples were stable when stored at-20 °C for a month and the processed samples had a good stability at 4 h and 24 h at room temperature.Pharmacokinetic parameters of AML in control group were as follows: AUC0-t: 64.40±25.54 ng/mL·h;AUC0-?: 74.58±29.81 ng/mL·h;t1/2: 6.91±1.64h;Tmax: 1.37±0.44h;Cl: 0.02±0.006 L/h/kg;MRT: 10.17±0.60;Cmax: 5.73±3.16 ng/mL.In contrast,pharmacokinetic parameters of AML in ginkgo leaf tablet group were as follows: AUC0-t: 33.49±10.70 ng/mL·h;AUC0-?: 36.72±11.06 ng/mL·h;t1/2: 6.70±1.07h;Tmax:2.50±1.30h;Cl: 0.03±0.008 L/h/kg;MRT: 10.26±1.43;Cmax: 3.29±1.34 ng/mL.Intriguingly,compared with the control group,there was a significant decrease in AUC0-t,AUC0-? and Cmax,accompanied with a significant increase in Tmax and Cl?P<0.05?in ginkgo leaf tablet group.There were 27 metabolites of AML detected with HPLC-Orbitrap-Fusion and Mass Frontier 7.0 software.Specifically,9 metabolites were detected in rat plasma of control group,including M2,M3,M5,M7,M14,M15,M16,M18 and M23,whereas 11 metabolites detected in rats of ginkgo leaf tablet group,including M2,M3,M5,M7,M9,M14,M16,M17,M18,M24,M22.More importantly,M15 and M23 were only detected in control group,accompanied with M17,M9,M22 and M24 detected only in ginkgo leaf tablet group.Conclusions: The UPLC-MS/MS method in detection of AML and its metabolites has a good performance in terms of accuracy,sensitivity and precision,which is,therefore,applicable for determination of the plasma concentration of AML in rats.The HPLC-Orbitrap-Fusion method established in our study has high resolution and fast analysis speed,therefore,27 kinds of metabolites have been detected.Thus,this method is preferable in the detection of the metabolites of AML in rat plasma.It has also been proved that combination of AML and ginkgo leaf tablet significantly inhibits the absorption of AML in rats,accelerates its removal and leads to the change of metabolite species.Part two: Selective inhibition of ethanol on muscarinic receptor-or 5-HT receptor-mediated contraction in the circular smooth muscle of rat stomachObjectives: To investigate the selective inhibition of ethanol on muscarinic receptor-or 5-HT receptor-mediated contractile responses in the circular smooth muscle strips isolated from the different regions of rat stomach.Methods: Circular muscle strips isolated from the rat gastric fundus,body,cardia and pylorus were prepared,and the contractile responses to carbachol?CCh?or 5-HT were recorded.Results: Ethanol?0.05‰0.5‰,v/v?did not affect the contractile response to CCh in circular muscle strips from the rat gastric fundus and cardia,and that to 5-HT in the strips from rat gastric fundus and body?P>0.05?.However,ethanol?0.1‰ and 0.5‰?significantly inhibited the Emax value of the contraction by CCh from 12.18±0.33 g of control level to 10.88±0.41 g and-LogEC50 value from 6.33±0.05 of control level to 6.12±0.06?P<0.05?in the strips from rat gastric body.Ethanol?0.1‰ and 0.5‰?also significantly inhibited the Emax value of the contraction by CCh from 2.87±0.15 g of control level to 2.2±0.13 g and-LogEC50 value from 6.49±0.10 of control level to 6.05±0.09?P<0.01?in the strips from rat gastric pylorus.Moreover,ethanol?0.1‰ and 0.5‰?significantly inhibited the Emax value of the contraction by 5-HT from 2.93±0.35 g of control level to 2.1±0.30 g?P<0.05?,but did not affect the-LogEC50 value in the strips from rat gastric cardia.Conclusions: Ethanol selectively inhibits the contractile responses to CCh or 5-HT in the circular muscle strips of rat stomach with a rank order of potency: gastric cardia?5-HT?> gastric pylorus?CCh?> gastric body?CCh?.In those gastric circular muscle strips,ethanol decreases both the activity and affinity of CCh to muscarinic receptors,but decreases only the activity of 5-HT to its receptors. |
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