白头翁皂苷主要活性成分的药代动力学研究
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摘要
白头翁为毛茛科植物白头翁Pulsatilla chinensis (Bunge) Regel的干燥根，为传统的清热解毒中药，现代药理学表明其具有抗肿瘤活性，本研究以主要活性部位白头翁皂苷为研究对象，对其化学成分、药动学、组织分布、排泄以及肠道部位的吸收和代谢进行了系统研究，为该中药的现代临床应用和开发提供依据。研究内容如下：
1、提取分离及含量测定
利用制备色谱，动态轴向色谱获得具有抗肿瘤活性的白头翁皂苷57g，再对该组分进行分离纯化获得5种成分，通过现代波谱技术鉴定了其结构，为齐墩果烷母核的白头翁皂苷，课题组命名为B3(70mg), BD (50mg), B7(70mg), B10(50mg)和B11(40mg)。经HPLC归一化法检查纯度均达到98%以上，可作为白头翁皂苷对照品使用。在此基础上，对白头翁皂苷进行含量测定，经HPLC-ELSD分析，B3，BD，B7， B10和B11的含量分别为24.1%，7.4%，12.4%，13.5%和8.5%。
2、药代动力学研究
首次建立了同时测定大鼠血浆中B3, BD, B7, B10, B11的RRLC-MS/MS法，以连翘苷为内标，采用ESI源负离子模式，多反应离子(MRM)监测进行分析。被测成分分别在1.105~13821(B3),0.7508~93.84(BD),0.9960~124.88(B7),0.4148~51.84(B10),0.3322~41.52(B11) ng·mL-1范围内线性关系良好，定量下限分别为1.105(B3),0.7508(BD),0.9960(B7),0.4148(B10)和0.3322(B11) ng·mL-1，待测物和内标的提取回收率均大于70%，方法的专属性、基质效应、精密度、准确度和稳定性均符合生物样品分析要求。采用所建立的方法测定了大鼠灌胃与静注两种给药途径下给予白头翁皂苷及其钠盐后5种主要成分的血浆浓度，获得了其在大鼠体内的药动学参数与绝对生物利用度。灌胃给药后的Tmax分别为0.33(B3),0.37(BD),0.51(B7,B10,B11)h,T1/2分别为12.81(B3),18.52(BD),16.31(B7),7.03(B10),12.54(B11) h。静脉给药后T1/2分别为0.43(B3),2.23(BD),0.56(B7),0.35(B10),0.34(B11) h。B3，BD，B7，B10和B11的绝对生物利用度分别为1.16%,1.17%,0.55%,0.96%和2.50%。结果表明：白头翁皂苷大鼠灌胃给药吸收迅速，消除较快，绝对生物利用度较低。
3、在体肠吸收研究
为了寻找白头翁皂苷口服相对生物利用度低的原因，建立了HPLC-ELSD法对其在大鼠体内的小肠吸收情况进行了考察。结果显示：5种白头翁皂苷中B3和BD的吸收系数（Ka），B3和B7的渗透系数(Peff)在不同肠段具有显著性差异（P<0.05）。5种白头翁皂苷的渗透系数(Peff)在不同肠段的吸收为十二指肠>空肠>结肠>回肠，十二指肠是该类皂苷的主要吸收部位。0.05~2.5mg·mL-1浓度范围内，5种白头翁皂苷随着浓度提高出现过饱和现象。该类皂苷在十二指肠的渗透系数(Peff)与浓度间的线性关系不明显(0.6007≤r2≤0.7727)，说明白头翁皂苷不完全依赖浓度梯度转运，细胞膜上的载体蛋白参与了药物的转运过程，其小肠吸收机制并不完全为被动转运。加入地高辛后，白头翁皂苷的渗透系数明显降低，而加入维拉帕米后，渗透系数明显提高，其中B3、BD、B7和B11有显著性差异(P<0.05)，说明白头翁皂苷为P-gp底物。
4、组织分布研究
以300mg·kg-1白头翁皂苷均匀分散混悬液单次灌胃给予大鼠后，对B3, BD, B7,B10, B11在动物体内的组织分布情况进行了考察。结果表明：白头翁皂苷在动物体内的分布迅速且广泛，给药15min后，各组织脏器中即可检测到较高水平的药物浓度。5种皂苷在大部分组织器官中的达峰时间为30min，给药2h后，白头翁皂苷的浓度逐渐下降，给药6h后，药物基本消除，进一步说明其在体内不易蓄积。药物主要分布在心脏，肝，肺，肾，小肠等器官，心脏中药物浓度最高。在动物脑中亦可检测到药物的存在，证明其可以透过血脑屏障。
5、排泄研究
以300mg·kg-1白头翁皂苷均匀分散悬浮液单次灌胃给予大鼠后，对B3, BD, B7,B10, B11原型药物的排泄情况进行了考察。主要研究白头翁皂苷在大鼠尿、粪和胆汁中的排泄量及累积排泄率。其结果显示给药后0~18h，其在雄性大鼠胆汁中的累积排泄量分别为12788.07(B3),2875.85(BD),2295.23(B7),1393.55(B10),635.46(B11) ng，分别相当于给药量的0.7075‰,0.5182‰,0.2468‰,0.1376‰和0.09968‰，存在微弱的肝肠循环；在给药后的0~108h，药物在大鼠尿液中的累积排泄量分别为3791.25(B3),539.24(BD),875.38(B7),495.55(B10),255.95(B11) ng，分别相当于给药量的0.2098‰,0.0972‰,0.0941‰,0.0489‰和0.0402‰；在给药后的0~108h，5种皂苷在大鼠粪便中的累积排泄量分别为5.791(B3),1.227(BD),3.194(B7),1.205(B10),1.042(B11) mg，分别相当于给药量的32.04%,22.10%,34.35%,11.90%和16.34%，研究结果提示大部分药物未被吸收，直接以原型形式由粪便排出体外；被动物机体吸收的部分，在体内大部分也已被代谢掉，以代谢产物的形式排出体外。
6、肠道代谢研究
考虑到该类成分的分子量较大，不符合“five rules”法则，属于较难吸收化合物，并且大部分齐墩果烷型五环三萜皂苷未以原型形式被机体吸收利用，为了寻找白头翁皂苷在较低的生物利用度下能产生确切的抗肿瘤活性的原因，需要进一步对该类皂苷在肠道的代谢情况进行研究。
6.1、肠道降解动力学
建立HPLC-ELSD法，采用肠道内容物体外孵育的方法，对白头翁皂苷的肠道降解动力学进行了考察。结果表明白头翁皂苷在大鼠肠道中孵育结果符合一级降解动力学特征，48h内白头翁皂苷B3, BD, B7, B10和B11迅速降解，降解速率常数(KA)为0.0794(B3),0.0523(BD),0.0539(B7),0.0426(B10)和0.0468(B11)，有效期(t0.9)为1.327(B3),2.015(BD),1.955(B7),2.473(B10)和2.251(B11) h，半衰期(t1/2)为8.730(B3),13.25(BD),12.86(B7),16.27(B10)和14.81(B11) h。肠道菌群是白头翁皂苷原型化合物发生代谢的重要因素。
6.2、肠道代谢产物初探
为进一步阐明白头翁皂苷在大鼠肠道菌群中发生降解后的代谢产物及代谢规律，采用UPLC-ESI-Q-TOF-MS/MS及Metabolite ID数据自动处理系统进行研究。结果显示，30小时内，正常大鼠肠内菌在实验条件下能有效代谢白头翁皂苷B3, BD, B7,B10, B11。实验发现8种B3代谢产物，7种BD代谢产物，8种B7代谢产物，7种B10代谢产物和9种B11代谢产物。白头翁皂苷在肠道的体外代谢产物共计40种。确证出该类成分在肠内菌群的作用下主要发生3-位侧链脱糖代谢，以及苷元母核上羟化，羧化和脱羧，甲基化和去甲基化等代谢反应。
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 determination
Fifty 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 research
A 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 absorption
In 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 distribution
The 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. Excretion
The 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 metabolism
Taking 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 dynamics
The 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.
1、提取分离及含量测定
利用制备色谱，动态轴向色谱获得具有抗肿瘤活性的白头翁皂苷57g，再对该组分进行分离纯化获得5种成分，通过现代波谱技术鉴定了其结构，为齐墩果烷母核的白头翁皂苷，课题组命名为B3(70mg), BD (50mg), B7(70mg), B10(50mg)和B11(40mg)。经HPLC归一化法检查纯度均达到98%以上，可作为白头翁皂苷对照品使用。在此基础上，对白头翁皂苷进行含量测定，经HPLC-ELSD分析，B3，BD，B7， B10和B11的含量分别为24.1%，7.4%，12.4%，13.5%和8.5%。
2、药代动力学研究
首次建立了同时测定大鼠血浆中B3, BD, B7, B10, B11的RRLC-MS/MS法，以连翘苷为内标，采用ESI源负离子模式，多反应离子(MRM)监测进行分析。被测成分分别在1.105~13821(B3),0.7508~93.84(BD),0.9960~124.88(B7),0.4148~51.84(B10),0.3322~41.52(B11) ng·mL-1范围内线性关系良好，定量下限分别为1.105(B3),0.7508(BD),0.9960(B7),0.4148(B10)和0.3322(B11) ng·mL-1，待测物和内标的提取回收率均大于70%，方法的专属性、基质效应、精密度、准确度和稳定性均符合生物样品分析要求。采用所建立的方法测定了大鼠灌胃与静注两种给药途径下给予白头翁皂苷及其钠盐后5种主要成分的血浆浓度，获得了其在大鼠体内的药动学参数与绝对生物利用度。灌胃给药后的Tmax分别为0.33(B3),0.37(BD),0.51(B7,B10,B11)h,T1/2分别为12.81(B3),18.52(BD),16.31(B7),7.03(B10),12.54(B11) h。静脉给药后T1/2分别为0.43(B3),2.23(BD),0.56(B7),0.35(B10),0.34(B11) h。B3，BD，B7，B10和B11的绝对生物利用度分别为1.16%,1.17%,0.55%,0.96%和2.50%。结果表明：白头翁皂苷大鼠灌胃给药吸收迅速，消除较快，绝对生物利用度较低。
3、在体肠吸收研究
为了寻找白头翁皂苷口服相对生物利用度低的原因，建立了HPLC-ELSD法对其在大鼠体内的小肠吸收情况进行了考察。结果显示：5种白头翁皂苷中B3和BD的吸收系数（Ka），B3和B7的渗透系数(Peff)在不同肠段具有显著性差异（P<0.05）。5种白头翁皂苷的渗透系数(Peff)在不同肠段的吸收为十二指肠>空肠>结肠>回肠，十二指肠是该类皂苷的主要吸收部位。0.05~2.5mg·mL-1浓度范围内，5种白头翁皂苷随着浓度提高出现过饱和现象。该类皂苷在十二指肠的渗透系数(Peff)与浓度间的线性关系不明显(0.6007≤r2≤0.7727)，说明白头翁皂苷不完全依赖浓度梯度转运，细胞膜上的载体蛋白参与了药物的转运过程，其小肠吸收机制并不完全为被动转运。加入地高辛后，白头翁皂苷的渗透系数明显降低，而加入维拉帕米后，渗透系数明显提高，其中B3、BD、B7和B11有显著性差异(P<0.05)，说明白头翁皂苷为P-gp底物。
4、组织分布研究
以300mg·kg-1白头翁皂苷均匀分散混悬液单次灌胃给予大鼠后，对B3, BD, B7,B10, B11在动物体内的组织分布情况进行了考察。结果表明：白头翁皂苷在动物体内的分布迅速且广泛，给药15min后，各组织脏器中即可检测到较高水平的药物浓度。5种皂苷在大部分组织器官中的达峰时间为30min，给药2h后，白头翁皂苷的浓度逐渐下降，给药6h后，药物基本消除，进一步说明其在体内不易蓄积。药物主要分布在心脏，肝，肺，肾，小肠等器官，心脏中药物浓度最高。在动物脑中亦可检测到药物的存在，证明其可以透过血脑屏障。
5、排泄研究
以300mg·kg-1白头翁皂苷均匀分散悬浮液单次灌胃给予大鼠后，对B3, BD, B7,B10, B11原型药物的排泄情况进行了考察。主要研究白头翁皂苷在大鼠尿、粪和胆汁中的排泄量及累积排泄率。其结果显示给药后0~18h，其在雄性大鼠胆汁中的累积排泄量分别为12788.07(B3),2875.85(BD),2295.23(B7),1393.55(B10),635.46(B11) ng，分别相当于给药量的0.7075‰,0.5182‰,0.2468‰,0.1376‰和0.09968‰，存在微弱的肝肠循环；在给药后的0~108h，药物在大鼠尿液中的累积排泄量分别为3791.25(B3),539.24(BD),875.38(B7),495.55(B10),255.95(B11) ng，分别相当于给药量的0.2098‰,0.0972‰,0.0941‰,0.0489‰和0.0402‰；在给药后的0~108h，5种皂苷在大鼠粪便中的累积排泄量分别为5.791(B3),1.227(BD),3.194(B7),1.205(B10),1.042(B11) mg，分别相当于给药量的32.04%,22.10%,34.35%,11.90%和16.34%，研究结果提示大部分药物未被吸收，直接以原型形式由粪便排出体外；被动物机体吸收的部分，在体内大部分也已被代谢掉，以代谢产物的形式排出体外。
6、肠道代谢研究
考虑到该类成分的分子量较大，不符合“five rules”法则，属于较难吸收化合物，并且大部分齐墩果烷型五环三萜皂苷未以原型形式被机体吸收利用，为了寻找白头翁皂苷在较低的生物利用度下能产生确切的抗肿瘤活性的原因，需要进一步对该类皂苷在肠道的代谢情况进行研究。
6.1、肠道降解动力学
建立HPLC-ELSD法，采用肠道内容物体外孵育的方法，对白头翁皂苷的肠道降解动力学进行了考察。结果表明白头翁皂苷在大鼠肠道中孵育结果符合一级降解动力学特征，48h内白头翁皂苷B3, BD, B7, B10和B11迅速降解，降解速率常数(KA)为0.0794(B3),0.0523(BD),0.0539(B7),0.0426(B10)和0.0468(B11)，有效期(t0.9)为1.327(B3),2.015(BD),1.955(B7),2.473(B10)和2.251(B11) h，半衰期(t1/2)为8.730(B3),13.25(BD),12.86(B7),16.27(B10)和14.81(B11) h。肠道菌群是白头翁皂苷原型化合物发生代谢的重要因素。
6.2、肠道代谢产物初探
为进一步阐明白头翁皂苷在大鼠肠道菌群中发生降解后的代谢产物及代谢规律，采用UPLC-ESI-Q-TOF-MS/MS及Metabolite ID数据自动处理系统进行研究。结果显示，30小时内，正常大鼠肠内菌在实验条件下能有效代谢白头翁皂苷B3, BD, B7,B10, B11。实验发现8种B3代谢产物，7种BD代谢产物，8种B7代谢产物，7种B10代谢产物和9种B11代谢产物。白头翁皂苷在肠道的体外代谢产物共计40种。确证出该类成分在肠内菌群的作用下主要发生3-位侧链脱糖代谢，以及苷元母核上羟化，羧化和脱羧，甲基化和去甲基化等代谢反应。
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 determination
Fifty 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 research
A 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 absorption
In 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 distribution
The 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. Excretion
The 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 metabolism
Taking 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 dynamics
The 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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