| Pulsatilla chinensis (Bunge) Regel, the dried root of its original botany, has been usedfor2000years as an antipyretic traditional oriental medicine. Taking the saponinscontained in Pulsatilla chinensis (Bunge) Regel (PRS) as research subjects which areresponsible for the antitumor activities, our research focused on their isolation andextraction, pharmacokinetics, absorption, tissue distribution, excretion and metabolism inthe biological system, so as to provide more evidences for further clinical applications anddevelopment.The major research content lists as follows:1. Extration, isolation and content determinationFifty seven gram of PRS which exerted potential antitumor activity has been extractedon preparative and dynamic axial chromatography. Then5oleanane pulchinenosides,which were named as B3(70mg), BD (50mg), B7(70mg), B10(50mg) and B11(40mg)by our team, has been isolated and purified from this PRS by various modern spectroscopictechniques and chemical methods.These components whose purities were all above98%determined by area normalization on HPLC could be used as reference substances for thecontent determination. Thereafter, the contents of B3, BD, B7, B10and B11in PRS weredetermined on HPLC-ELSD as24.1%,7.4%,12.4%,13.5%and8.5%, respectively.2. Pharmacokinetic researchA simple, RRLC-ESI-MS/MS method was developed for the simultaneousdetermination of five oleanane pulchinenosides (B3, BD, B7, B10and B11) in rat plasmafor the first time. Applying forsythin as internal standard (IS), detection and quantitationwere performed by MS/MS using electrospray ionization (ESI) and multiple reactionmonitoring (MRM) mode. The5analytes showed good linearity for1.105~13821(B3),0.7508~93.84(BD),0.9960~124.88(B7),0.4148~51.84(B10),0.3322~41.52(B11)ng·mL-1, and the LLOQs for B3,BD,B7,B10and B11were1.105(B3),0.7508(BD),0.9960(B7),0.4148(B10) and0.3322(B11) ng·mL-1. Meanwhile, the recoveries of the analytes and IS were all above70%. The specificities, matrix effects, precisions, accuraciesand stabilities of the5components were validated in accordance with the guidance for thebioanalytical methods validation. The plasma concentrations of the5analytes for oral andintravenous administration in rat could be determined by the established method, then thepharmacokinetics parameters and bioavailabilities were obtained. After oral administration,Tmaxwere0.33(B3),0.37(BD),0.51(B7, B10, B11) h, respectively; T1/2were12.81(B3),18.52(BD),16.31(B7),7.03(B10),12.54(B11) h, respectively. After intravenous, T1/2were0.43(B3),2.23(BD),0.56(B7),0.35(B10),0.34(B11) h. The bioavailabilities of B3,BD, B7, B10and B11were1.16%,1.17%,0.55%,0.96%and2.50%, respectively. Theresults indicated that PRS exhibited rapid absorption and were eliminated quicklythereafter for oral administration. Therefor, the bioavailability were relatively lower.3. Intestinal absorptionIn order to search for the reason why the bioavailabilities were relatively lower,HPLC-ELSD was applied to explore the absorption mechanism of pulchinenosides (B3,BD, B7, B10, B11) in rats. The Ka value (B3,BD) and Peffvalue (B3,B7) displayedsignificant difference(P<0.05) in various intestinal segments.The Ka value and Peffvalue ofPRS was different from each other with the highest absorption in duodenum (duodenum>jejunum> colon> ileum), so the duodenum was the main absorption site.The PRSdisplayed excessive satuation as the concentration increased over0.05-2.5mg·mL-1. Therewere no obvious linear correlations between Peffvalues and concentrations in duodenum(0.6007≤r2≤0.7727), which demonstrated PRS didn’t entirely transported in aconcentration dependent manner, and the transporter-protein involved the transportation,so the intestinal absorption of the five pulchinenosides was not entirely passive diffusion.The Ka value and Peffvalue declined when the PRS was perfused with P-glycoproteinpromoter digoxin, on the other hand, inclined when perfused with P-glycoprotein inhibitorverapamil with significant difference among PRS B3, BD, B7, B11(P<0.05), whichmanifested that the PRS might be the substrates of P-glycoprotein.4. Tissue distributionThe distribution of pulchinenosides (B3, BD, B7, B10, B11) in rats tissue has beeninvestigated after300mg·kg-1oral administration of PRS evenly dispersed suspension. Theresults suggested that PRS exhibited rapid and extensive distribution, and high levelconcentrations could be detected in rats’ tissues after0.25h for oral administration. Most of the Tmaxof5components in the tissues were about30min, then the concentrationsdeclined gradually after2h and a large proportion of PRS were almost eliminated after6h,which further indicated that PRS were difficult to accumulate in vivo. The medicine mainlydistributed in heart, liver, lungs, spleen and small intestine, and it was highest in the heartthan in other tissues. We can also detected PRS in the brain, which could demonstrated thatPRS could transmitted across blood-brain barrier (BBB).5. ExcretionThe excretion of pulchinenosides (B3, BD, B7, B10, B11) in rats has beeninvestigated after300mg·kg-1oral administration of PRS evenly dispersed suspension. Theexcretion amounts and accumulative excretion rates in rats’ bile, urine and feces have beendetermined. The result showed that the accumulative excretions of PRS in male rats’ bilein0-18h after oral administration were12788.07(B3),2875.85(BD),2295.23(B7),1393.55(B10),635.46(B11) ng, equivalently0.7075‰,0.5182‰,0.2468‰,0.1376‰and0.09968‰to the dosage, respectively, which demonstrated there was slightlyenterohepatic circulation in rats; the accumulative excretions of PRS in male rats’ urine in0-108h after oral administration were3791.25(B3),539.24(BD),875.38(B7),495.55(B10),255.95(B11) ng, equivalently0.2098‰,0.0972‰,0.0941‰,0.0489‰and0.0402‰to the dosage, respectively; the accumulative excretions of PRS in male rats’feces in0-108h after oral administration were5.791(B3),1.227(BD),3.194(B7),1.205(B10),1.042(B11) mg, equivalently32.04%,22.10%,34.35%,11.90%and16.34%to thedosage, respectively. The result suggested that a large proportion of PRS have not beenabsorbed and excreted with feces out of the body as the prototypes; the proportion whichwere absorbed in the body also have been metabolized and excreted with feces out of thebody as the metabolite types.6. Intestinal metabolismTaking the factors into consideration that the molecular weight of PRS were relativelyhigher so as not to accord with “five rules”, and were categorized as “hard to be absorbed”compounds, Meanwhile, most of which were not absorbed and utilized as the prototypesby the organism, it is necessary to further explore the metabolism of PRS in intestine tosearch for the reason why the bioavailabilities of PRS were relatively lower but exertedsignificant antitumor activity. 6.1. Intestinal degradation dynamicsThe method of incubation with rat intestinal bacteria in vivo was applied to researchthe degradation dynamics of PRS on HPLC-ELSD. The result showed that PRS were inaccordance with first-order reaction with rapid degradation in rat intestinal flora declinedin48hours, and the degradation rate constants (KA) were0.0794(B3),0.0523(BD),0.0539(B7),0.0426(B10) and0.0468(B11), respectively; the t0.9were1.327(B3),2.015(BD),1.955(B7),2.473(B10) and2.251(B11) h, respectively; the t1/2were8.730(B3),13.25(BD),12.86(B7),16.27(B10) and14.81(B11) h, respectively. Therefor, ratintestinal flora was the most important factor on the metabolism of prototype compoundsof PRS under experimental conditions.6.2. The primary exploration of metabolites in rat intestinal flora.The metabolites of pulchinenosides B3, BD, B7, B10and B11in rat intestinal florahave been researched on UPLC-ESI-Q-TOF-MS/MS and Metabolite ID data processingsystem. The result was that normal rat intestinal flora could metabolite B3, BD, B7, B10,B11effectively under experimental conditions in30hours.8metabolites of B3,7metaboites of BD,8metaboites of B7,7metaboites of B10,9metaboites of B11have beendetected in rat intestinal bacteria, that was totally40metabolites. A variety of metabolitetypes have been concluded in intestinal bacteria mainly such as3-side chain sugar-remove,hydroxylation, carboxylation and decarboxylation, methylation and demethylation on theparent nucleus of glycoside. |
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