Studies On Isolation And Pharmacokinetics Of Chemical Constituents From Trollius Chinensis

Jinlianhua, dried flowers of Trollius chinensis Bunge, has been used as an important traditional Chinese medicine (TCM) in Chinese folk for a long history.《Bencao Gangmu Shiyi》said that it was"bitter in taste, cold-natured,asepsis",and could be used for the treatment of aphtha,larynx boss,light fever atrophy of the gum,ear ache,ophthalmalgia",and that it had the effects of eyesight improvement and anti-miasma. Modern pharmacological studies suggest that it possesses antimicrobial and antiviral actions and has been used widely to treat cold, fever, chronic tonsillitis,acute tympanitis,urinary tract infection and other inflammations. Previous studies demonstrated that trollius mianly contains flavonoids and organic acid components, the aqueous extract of which has a strong antiviral action, and its main active compenents are flavonoids.With the help of dynamic and kinetic mechanisms and mathematics method, pharmacokinetics of traditional Chinese medicine (TCM),under the guide of chinese medical science theory, studied absorption, distribution, biotransformation and elimination of active constituents or parts, simple or compound recipes of TCM, and relations of pharmacokinetics and pharmacodynamics of TCM. As one of important contents of modernization of TCM, pharmacokinetics of TCM might be combine the studies of phytochemistry and pharmacodynamics and were beneficial for illuminating efficacy of TCM, identifying therapeutical basis and mechanism of action of TCM.In the present study, a separation method of orientin-2"-O -β-L-galactopyranosyl(OGA), orientin and vitexin from Trollius chinensis was established. Pharmcokinetic of OGA, orientin and vitexin after intravonous administration of Trollius chinensis extract (TCE) and pharmcokinetic and tissue distribution of orientin after intravonous administration of oreintin monomer to rats were investigated for illuminating mechanism of action of theirs. The research provided a significant exploration for pharmacokinetics and therapeutic basis of TCM.Part 1 Study on chemical constituents of Trollius chinensis (Preparation of orientin-2′′-O-β-L-galactopyranosyl, orientin and vitexin)Objective: To establish a separation method of orientin-2′′-O-β-L-galactopyranosyl, orientin and vitexin from Trollius chinensi.Methods: After extracted with alcohol and precipitated with hot water from Trollius chinensi, the extract was isolated and purified by silica gel column chromatography and preparative HPLC. Orientin-2′′-O-β-L-galactopyranosyl, orientin and vitexin were identified by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC).Results: Three compounds were separated from Trollius chinensi and identified as: Orientin-2′′-O-β-L-galactopyranosyl, orientin and vitexin. The purity of orientin-2′′-O-β-L-galactopyr -anosyl, orientin and vitexin than 98% by normalization method of HPLC.Conclusion: The developed method is simple and at low production cost. The products can be used as reference substance for quality control and drugs for pharmacokinetic study.Part 2 Studies on pharmacokinetics of three active flavonoids in rats after intravenous administration of Trollius chinensis extractObjective: To develople a HPLC method for simultaneous determination of orientin-2′′-O-β-L-galactopyranosyl, orientin and vitexin in rat plasma for studying the pharmacokinetics of them after a single intravenous administration of Trollius chinensis extract.Methods: After rats were intravenous administrated extract solution at a dose of 4mL·kg^-1, blood samples were obtained from fossa orbitalis vein according to the specific schedule, 2, 5, 10, 15, 20, 30, 40, 50, 60, 90, 120 and 180min and collected in heparinized centrifuge tube, respectively. Sample was pretreated by a single-step protein precipitation with methanol. The RP-C18 column (250×4.6mm,5μm)was used as the stationary phase with the gradient mobile phase consisting of methanol and 0.1% acetic water. The flow rate was 1mL·min-1, the UV detector was set at 348nm.Results: Calibration curves of orientin-2′′-O-β-L-galactop -yranosyl, orientin and vitexin were generated over the range 0.315¹61, 0.326¹67 and 0.215¹10μg/mL, respectively, and the LOQ of them were 0.04, 0.03 and 0.03μg/mL, respectively. The intra- and inter-day precisions (RSD) for the analysis of three analytes were between 1.68% and 8.43% with accuracies (RE) below 8.55%. The mean extraction recoveries were between 70.35% and 86.42%. After single intravenous administration of 4mL·kg^-1 extract solution of Trollius chinensis to rats, the main pharmacokinetic parameters of orientin-2′′- O-β-L-galactopyranosyl, orientin and vitexin were as follows: t1/2αwere 1.72±0.58, 1.88±0.23 and 1.22±0.42min, t1/2βwere 10.38±5.75, 11.88±0.46 and 9.99±1.05min,t1/2γwere 36.88±3.71, 64.50±9.22 and 45.64±7.66min,V(c)were 0.018±0.007, 0.016±0.001 and 0.015±0.006L·kg^-1, CL(S)were 0.002±0.000, 0.002±0.000 and 0.003±0.000L·(kg·min-1,AUC(0-t)were 3539.4±103.9, 2840.8±215.6 and 581.5±59.5mg·min·L^-1,MRT(0-t)were 31.46±0.88, 15.86±0.59 and 14.25±0.48min。Conclusion: A simple HPLC assay method has been developed for simultaneous determination of orientin-2′′-O-β-L- galactopyranosyl, orientin and vitexin in rat plasma following an intravenous administration of the extract of Trollius chinensis and the method was was successfully applied to the pharmacokinetic studies of the three active components. The method with high sensitivity, specificity, good precision and accuracy is fit to the needs of biosample analysis.Part 3 Studies on pharmacokinetic and tissue distribution of orientin in ratsObjective: 1. A HPLC method for determination of orientin in rat plasma was developed for studying the pharmacokinetics of orientin in rats after a single intravenous administration of orientin at three different dosages. 2. A HPLC method was developed and validated for determination of orientin in rat tissues.Methods: 1. After rats were intravenous administrated orientin, blood samples were obtained from fossa orbitalis vein according to the specific schedule, 2,4,8,12,16,20,25,30,45,60 and 90min and collected in heparinized centrifuge tube, respectively. Sample was pretreated by protein precipitation with methanol using puerarin as internal standar (IS),the supernatant was collected and evaporated to dryness at 40℃under a gentle stream of nitrogen. The residue was then reconstituted with 100μL mobile phase, and centrifuged at 12000×g for 5 min, and an aliquot (20μL) of the supernatant was injected into the HPLC system. The RP-C18 column (250×4.6mm,5μm) was used as the stationary phase with the mobile phase consisting of acetonitrile–0.1% acetic water (20:80, v/v) at a flow rate of 1.0mL·min-1. Chromatograms were monitored at 250nm for IS, 348nm for orientin and the temperature of column was kept at 35℃. The flow rate was 1mL·min-1. 2. Rats were randomly assigned to six groups. After intravenous administration of 20mg·kg^-1 solution of orientin, heart, liver, lung, spleen, kidney, brain, stomach and small intestine samples were obtained at 5, 15, 30, 45, 60 and 90min, respectively. Tissue samples were weighed rapidly and put into normal saline solution to remove the blood or content, blotted on filter paper, and then were weighed for wet weight and homogenized in saline solution (500mg·mL^-1). Preparation of tissues samples and HPLC analysis conditon were same with the plasma samples, except that IS was not used. The method was applied to study tissues distribution of oreintin in rats after a single administration of orientin at a dose of 20mg·kg^-1.Results: 1. The calibration curve in plasma was linear over the range of 0.250⁵0.1μg·mL^-1 and the RSD values of intra-day and inter-day were less than 15%. The recovery of orientin in rat plasma was 68.0%⁷3.1%. The study of stability demonstrated samples were stable. After single intravenous of three different dosages 10, 20 and 30mg·kg^-1 orientin to rats, the main pharmacokinetic parameters were as follows: V(c) were 0.364±0.085, 0.233±0.014 and 0.259±0.023L·kg^-1, CL(S) were 0.057±0.022, 0.035±0.020 and 0.049±0.003L·(kg·min)-1, T1/2γwere 14.34±2.16, 22.53±3.48 and 25.29±3.27min,AUC0-t were 150.601±11.528, 422.392±54.063 and 593.473±25.516μg·min·mL^-1. 2. The intra-day and inter-day presion were less than 15%, and the extraction recoveries of different tissues were larger than 65%. After a single administration of orietin at a dose of 20mg·kg^-1, the highest level was observed in kidney, then in liver and lung. The lowest level was found in brain.Conclusion: 1. Orientin was eliminated rapidly in rat and manifested linear dynamics at 10³0mg·kg^-1 range. 2. Kidney,liver and lung were the main distribution tissues of orientin in rats, and orientin had difficulties in crossing the blood brain barrier. It was also found there was no accumulation of loganin in rat tissues.