The Pharmacokinetic Study Of Three Phenylethanoid Glycosides In Callicarpa Nudflora

ObjectiveCallicarpa nudflora Hook.et Am.,belonged to Verbenaceae genus,is the famous region drug in Hainan province.It was first recorded in 1977 Hainan floras of material medica.It and its tablets have the actions in haemostasis,antiinflammation,bacteriostasis and so on.It is commonly used in the therapy of haemostasis and antiinflammation in clinic.But the pharmacokinetic studies were also rarely reports.Therefore,this paper selected the main effective components groups,the phenylethanoid glycosides(PhGs),to measure the contents in different herbs samples(different medicinal parts,growth periods and fresh/dry samples).Measure the contents in herbs,extracts and tablets were to study the effect of the PhGs during tablets making process.Determination the concentrations of three PhGs after intragastric administration of the tablets were to study their kinetics process,main PK parameters and tissues distribution in rats.Methods1.The three PhGs compounds were separated and purified by macroporous resin,ODS and sephadex column chromatography technology.The structures were identificated by MS,13C-NMR,1H-NMR and so on.The purity was measured by TLC with three different thin layer systems and HPLC.2.The HPLC method for determination of three PhGs component content in C.nudiflor herbs,extracts and tablets was established.The instrument was Agilent 1260 HPLC,the chromatographic column was C18 column,the mobile phase was methanol-0.1%formic acid solution(40:60),the detection wavelength was 334 nm.3.The LC-MS/MS method for determination of three PhGs component content in biological samples after intragastric administration of C.nudflora tablets in rats was established.The instrument was Agilent 1260-6460 LC-MS/MS,the chromatographic column was Phenomenex Luna 5um C18(2)100A 150 mm×2.0 mm,the column,the mobile phase system was acetonitrile/0.1%formic acid gradient elution.The ion source of mass spectrometry was ESI+Agilent Jet S,the gas temp was 300 ?,the gas flow was 5 1/min,the nebulizer pressure was 45 psi,the sheath gas temp was 300 ?,the sheath gas flow was 11 1/min,the capillary was 3500 V,the nozzle voltage was 500 V.The MS/MS ion transitions monitored were m/z 755?161(forsythiaside B,49 eV),623?161(acteoside,33 eV),623?161(isoacteoside,37 eV),163?119(coumaric acid,13e V).4.The method for making biological samples:100 ?l rat plasma(or tissues slurry),added 10 ?l courmaric acid,vortexed oscillation for 3 min,added 290 ?l methanol,vortexed oscillation for 5 min,centrifugal at 4? and 12000r/min for 15 min,did the same one more time,took the supernatant fluid sample for analysis.Results1.The isolation of forsythiaside B was 736 mg,acteoside was 129 mg and isoacteoside was 118 mg.The purity of each one is more than 98%.2.The contents of forsythiaside B was 0.02?10.47 mg/g,acteoside was 0.06?65.43 mg/g,isoacteoside was 0.01?6.70 mg/g.The contents of three PhGs were higher in leaves than in branches,higher in fresh samples than in dry samples.The contents of three PhGs in leaves that planted for one and two years were higher than the eight years',which should be noticed for collecting.In C.nudiflor herb,the extraction rate was 30%,the mean contents of three PhGs were 5.26 mg/g,29.75 mg/g and 4.94 mg/g.In extract,the contents of three PhGs were 31.49 mg/g,20.22 mg/g and 24.45 mg/g.The transfer rates were 55.67%,20.39%,67.36%from herb to extract,which showed that the stability of acteoside was poor and the transfer rates of forsythiaside B and isoacteoside were high after extraction.In tablets,the mean contents of three PhGs were 30.73 mg/g,17.09 mg/g and 23.87 mg/g.The transfer rates were 2.38%,15.48%,2.37%from extract to tablet,which showed that the tablet making process was stable.3.The concentration of three PhGs in plasma samples were determinied by LC-MS/MS and the main PK parameters were gained.The C-T curves were drawed by the concentration-time datas.The C-T curves of plasma,heart,spleen,lung,kidney,brain,muscle,ovary,testis and stomach showed bimodal phenomenon.The Tmax in the three PhGs were 0.50 h,0.75 h,0.50 h,meaning that these compounds were quickly absorbed from the gastrointestinal tract.The elimination was slow after intragastric administration(t1/2 in three PhGs were 5.57 h,7.26 h,4.09 h).Three PhGs compounds were widely distributed in the tissues in rats.The concentrations were high in stomach and duodenum,meaning that the metabolism was mainly in gastrointestinal tract.They could through the blood brain barrier and blood testosterone barrier.The three PhGs were measurd in ovary,meaning that it was related to the tablet's function of treating gynaecology diseases.ConclusionDetermination the concentration of three PhGs in C.nudiflor herbs was to control the quality of the herb.Determination the concentration of three PhGs in herbs,extracts and tablets was to provide the basis for tablet's making process.The kinetics process of these PhGs in rats was fit to noncompartmental model after intragastric administration C.nudiflor tablets.The absorption of these compounds was fast and the elimination was slow.These PhGs were widely distributed and were high in stomach and duodenum and could through the blood brain barrier and blood testosterone barrier.