Studies On Pharmacokinetics Of Bergenin In Healthy Chinese Volunteers/Mice And Effects Of Bergenin On I_GABA

Objectives:To firstly establish highly sensitive, simple and selective high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methods for the determination of bergenin in plasma after oral administration of bergenin tablets.To study the pharmacokinetics and tissue distribution. of bergenin in healthy mice after 3mg-kg"'bergenin intravenous injection by HPLC, to evaluate its pharmacokinetic parameters and rule.To apply HPLC-MS/MS method to the determination of bergenin concentration in human plasma after single dose (250mg) oral administration.To establish the HPLC-MS method for determine the low concentration of chlorpheniramine in human plasma after single dose(4mg) oral administration in complex tablets consists of bergenin and chlorpheniramine.To investigate the modulatory effect of bergenin onγ-aminobutyric acid (GABA)activated currents(IGABA)-patch clamp technique was performed on rat hippocampal pyramidal neurons and HEK293T cell (transfected of GABAA receptor)Methods:.Bergenin (1) Chromatographic conditions:A C18 column (50mm×2.1mm,3.5μm) (Agilent, U.S.A) was used for chromatographic separations. Bergenin and I.S. were eluted with mobile phase of acetonitrile-water(10:90, V/V)at a flow-rate of 0.2 mL/min. The total run time for a sample was about 6.5 min. The detection occurred at 25℃. (2) Mass spectrometric conditions:The ion source was set at the negative ionization mode (ESI+) and ion collection was occurred at the multi-reaction monitoring (MRM) mode, nitrogen was used as collision gas (with a collision energy of 15V for bergenin and 30V for thiamphenicol). Nebulizer gas was set at 40 psi, and spray gas at 9L-min-1. The temperature of the turbo ion spray source was 350℃. The capillary voltage was 4000V. The transitions of m/z 327.1^192 for bergenin and 354→185.1 for thiamphenicol (I.S.) were selected as quantification signals respectively. The scan time for bergenin was set at 0.4s.Chlorpheniramine (1)Chromatographic conditions:The separation was performed on a Venusil XBP-C18column (5μm, ID4.6×150mm), and eluted by the mobile phase consisted of a methanol-0.005M ammonia acetate solution (60:40, v/v) at a flow rate of 0.8mL/min. The column temperature was maintained at 25℃using a thermostated column compartment. (2)Mass spectrometric conditions:Mass spectrometric analysis was performed in the ESI positive ion mode, with spray gas pressure of 350 Pa, protective air of nitrogen gas at a flow rate of 13 L-min-1, capillary voltage of 4000 V, fragment electric voltage of 90 V for chlorpheniramine and 70 V for diphenhydramine. Full scan mode was performed for screening and library-assisted identification. The pseudomolecular ions [M-H]ˉ(m/z 275.1 for Chlorpheniramine and 256.1 for diphenhydramine) were used as the target ions for quantification in the selected ion monitoring (SIM) mode.Sample preparation:(1)Bergenin:0.5 mL plasma and 20μL I.S. solution (0.4μg·mL-1) were added to centrifuge tube containing 4 mL acetic ether, vortex-mixed for 2 min, then centrifuged at 5000 rpm (revolutions per minute) for 5 min.3.5 mL supernatant was transferred and evaporated to dryness under gentle stream of nitrogen in water-bath at 40℃. The residue was reconstituted with 100μL of acetonitrile-water (10:90, V/V) and 20μL supernatant was injected for analysis. Blank plasma samples, calibration series, QC and plasma samples were analyzed in one analytical sequence. (2)Chlorpheniramine:50μLof the internal standard (200 ng·mL-1 diphenhydramine) was mixed with 0.5 mL plasma sample, then 5mL methylene chloride were added, vortex-mixed for 2 min, and centrifuged at 4000 rpm for 5 min. The organic phase was transferred to a clean tube and evaporated to dryness under gentle nitrogen gas at 45℃. The residue was reconstituted with methanol-0.005M ammonia acetate solution (60:40, v/v), and 10μL was injected onto the LC/MS for analysis.Twenty healthy Chinese volunteers were orally given 254 mg complex tablets consists of 250 mg bergenin and 4mg chlorpheniramine with 200 mL water in the morning and 4 mL of blood were collected before (0 h) and 0.5,1,1.5,2.0,3.0,4.0,5.0, 6.0,8.0,12.0 24.0and 36.0 h after administrations, serum were separated, collected and stored at-20℃for analysis. The main pharmacokinetic parameters were calculated by Drug and Statistic software (DAS, version 2.0.1, by Sun et al, China).Healthy mice plasma were collected from vein or decollated for dissection of liver, spleen, kidney, intestine, lung, heart and brain after introvenous injection of 3 mg·kg-1 bergenin. Use HPLC to study the pharmacokinetics and tissue distribution, of bergenin in healthy mice after 3mg·kg-1 bergenin intravenous injection.Whole-cell configuration of patch clamp technique and outside-out configuration for single channal recording were performed on rat hippocampal pyramidal neurons and HEK293T cell (transfectde of GABAA receptor)to investigate the modulatory effect of bergenin on y-aminobutyric acid(GABA)-activated currents (IGABA). I-V curve and dose-response of Bergenin were recorded in the experiment. Glutamate activated currents (IGlu) was also observed to study the potential antitussive mechanism of bergenin in central nervous system.Results:It is proved that the HPLC-MS/MS methods is highly sensitive, simple and selective. The linear range of the calibration curve for determination of bergenin in plasma by the method was 0.25-60 ng·mL-1, and the low limit of quantitation (LOQ) was 0.25 ng·mL-1. The main pharmacokinetic parameters of bergenin after single oral doses in human plasma were as follows:t1/2 2.850±1.823h, Tmax 1.725±0.638h, AUC0～12 49.013±14.13ng·ml-1·h, Cmax 15.216±5.023ng·ml-1, AUC0～∞51.352±15.194ng·ml-1·h.The linear range of the calibration curve for determination of chlorpheniramine in human plasma after oral administration of complex tablets consists of 250 mg bergenin and 4mg chlorpheniramine by the HPLC-MS method was 0.2-20 ng·mL-1, and the low limit of quantitation was 0.2ng·mL-1. The main pharmacokinetic parameters of bergenin after single oral doses in human plasma were as follows:Tmax 2.650±0.763h, t1/2 11.480±3.605h, AUC0～36 98.636±22.863ng·ml-1·h, AUC0～∞113.173±32.390ng·ml-1·h, Cmax 6.636±1.533ng·ml-1。The main pharmacokinetic parameters of bergenin after intravenous injection (3mg·kg-1) in mice plasma were as follows:t1/2 1.16±0.03 h, Cmax 2443.3±557.6 mg·L-1, AUC0-8 2482.6+281.4 mg·ml-1·h, AUC0-∞2482.9±331.0 mg·ml-1·h, Vd 1.07±0.08L·kg-1。Bergenin was distributed widely in mice,the concentration in liver, and kidney is higher than others..The concentration of bergenin in brain is relatively higher than the concentration in lung.Bergenin was declined quickly in the body.The results showed that GABA-activated membrane currents were obviously increased by bergenin. The GABA-activated current (IGABA)was inhibited by bicuculline(10μmol·L-1) that showed the current was mediated by GABAA receptor. Dose-response relationship of bergenin(0.02,0.1,0.5,1,5,10mmol·L-1) on IGABA shows IGABA can be increased by bergenin within 0.02-10mmol/L.The dose response curve is "s" shape and EC50 is 0.81mmol·L-1. Bergenin shifted the GAB A dose-response curve upward,however, the maximum response keeps unchanged. The single channel activity was abolished by 10μmol·L-1 bicuculline confirming it was mediated by GABAA receptors. Bergenin did not induce any detectable single channel activity on its own. Results suggest that the effect of bergenin on GABAA receptors was achieved by affecting channel open probability, not channel conductance. IGLU keeped unchanged when bergenin was in or not.Conclusions: The firstly establishde HPLC-MS/MS methods is highly sensitive, simple and selective. The linear range of the calibration curve for determination of bergenin in plasma by the method is 0.25-60 ng·mL-1, and LOQ is 0.25 ng·mL-1. Bergenin is absorbed into the plasma rapidly not completely, and the short t1/2 showed that elimination of bergenin is quick.The HPLC-MS method developed in this study is suitable to meet the requirement of determination of low chlorpheniramine concentration in human plasma after its therapeutic oral doses in complex tablets consists of bergenin (125mg) and chlorpheniramine (2mg). The linear range of the calibration curve for determination of chlorpheniramine in plasma by the method is 0.2-20 ng·mL-1, and LOQ is 0.2 ng·mL-1. chlorpheniramine is absorbed into the plasma rapidly and completely. The t1/2 of chlorpheniramine is longer than bergenin shows that chlorpheniramin can be storaged in human body for a longer time.Pharmacokinetics and tissue distribution characteristics of bergenin in mice shows that bergenin is distributed widely and eliminated rapidly. Besides, the concentration of bergenin in brain is relatively higher than the concentration in lung shows that antitussive action of bergenin may be related with central nervous system.Bergenin can increase IGABA but it cannot activate current without GABA. The effect of bergenin on GABAA receptors was achieved by affecting channel open probability, not channel conductance.Bergenin has no effect on IGlu-It is the first report about possible central antitussive mechanism of bergenin.No adverse reactions of bergenin and chlorpheniramine were observed in the study.