【作者】 潘洪平；

【导师】 李高；

【作者基本信息】 华中科技大学 ， 药理学， 2010， 博士

【摘要】 葛根素是一种多羟基黄酮类化合物,是临床治疗心脑血管病的常用药物之一。葛根素是P-gp的底物,这是造成其跨膜透过性低的重要原因之一。该药物口服吸收差,目前主要通过静脉注射给药,但葛根素注射剂具有较为严重的不良反应,临床应用受限。为了解决其口服生物利用度低的问题,有必要对P-gp抑制剂促进葛根素口服吸收的生物因素进行研究。但是传统的P-gp抑制剂如普罗帕酮、维拉帕米等具有严重的毒副作用(如心脏毒性),为寻求高效、低毒的P-gp抑制剂和有效地提高葛根素的口服吸收利用率,本论文对槲皮素(一种葛根素的结构类似物和P-gp抑制剂)影响葛根素的肠道吸收作用进行了系统研究。此外,还考察了其对葛根素跨膜分布于脑组织的影响,并初步探讨了葛根素保护缺血脑损伤的作用机制。利用大鼠离体肠囊外翻模型,观察槲皮素对葛根素从外翻肠囊黏膜侧到浆膜侧跨膜转运量的变化(吸收作用)和由浆膜侧向黏膜侧进行转运的量变过程(外排作用),并对组间计量资料进行统计学处理和分析。结果发现：①在缺乏P-gp抑制剂影响下,十二指肠、空肠和回肠段对葛根素的透膜吸收作用无明显差异,由于回肠的P-gp表达量最多,故选择回肠段作为评价不同P-gp抑制剂对小肠吸收葛根素和外排葛根素的影响；②吸收试验表明,维拉帕米和槲皮素于药后10min均可明显提高单位面积的葛根素累积吸收量(P<0.01),且各药物组对葛根素的吸收量均随时间延长而明显增加,作用一直延至药后120min。槲皮素高剂量对葛根素的透膜吸收作用明显强于阳性组维拉帕米,药后40min—120min两组比较均有显著意义(P<0.05),槲皮素对葛根素的促吸收作用具有一定的量效关系；③外排试验表明,从0-120min各组的浆膜侧葛根素浓度均随时间延长而明显减少,药后30min各药物组在各取样时间点均可明显增加浆膜侧的葛根素含量,抑制P-gp120min后,阳性组对单位面积葛根素外排剩余量提高率为138.79%,槲皮素高、中、低剂量组的提高率分别为219.06%、107.29%和60.43%。槲皮素高剂量的外排抑制作用强于维拉帕米(P<0.01),槲皮素对葛根素的外排抑制作用具有一定的量效关系；④葡萄糖吸收试验表明,0→120min内,肠浆膜侧内液具有明显的逆浓度差聚集葡萄糖的作用,均达3倍以上,证明所用的实验肠段具备组织完整性和生理活性。上述研究结果表明,槲皮素具有通过抑制P-gp而促进葛根素的肠道吸收作用。采用大鼠在体回肠单向灌流模型和重量法,进一步计算和表征葛根素的吸收百分率Pa%、吸收速率常数Ka值和表观吸收系数Peff值等相关肠道吸收速率和渗透性参数,结果发现：①葛根素在肠灌流液中6h内保持良好的稳定性,提示实验时程内进行葛根素含量测定其质量稳定可控；②各实验组的灌流出口液葛根素浓度与灌流进口液葛根素浓度之比(Cout／Cin)在取样的135min内基本保持一致,各组的组内净水流量NWF值均无显著差异,提示取样前的灌流平衡时间可使肠道的药物吸收及水分吸收达到平衡状态；③维拉帕米和槲皮素均可显著提高Ka、Peff和Pa%值(P<0.01),槲皮素高剂量的作用强于维拉帕米(P<0.01),槲皮素的作用具有一定的量效关系；④根据灌流液校正密度计算所得的Pa%、Ka、Peff值对于低吸收率药物,影响较大,为减少实验结果的系统误差,有必要对灌流进出液的密度进行修正。上述研究结果表明,槲皮素具有通过抑制P-gp而提高葛根素的肠道吸收速率和渗透性的作用。以小鼠整体动物实验结合前述的体外法和原位实验模型法对葛根素的肠道吸收作用进行初步体内评价。结果发现：①灌胃给予葛根素后其血药浓度经时变化规律符合二室模型,t1／2Ka=5.565±5.094min,Ka=0.152±0.084min-1；②与阴性组比较,维拉帕米和槲皮素组均具有明显增大吸收分数F值和显著增加AUC值的作用,分别将AUC(0-t)提高了33.03%和156.46%,将AUC(0-∞)提高了37.04%和164.27%,还分别将AUC(0-Tmax)提高了38.57%和110.28%,槲皮素组的AUC(0-t)、AUC(0-∞)和AUC(0-Tmax)均明显高于维拉帕米组(P<0.01)；③槲皮素组对MRT(0-t)和MRT(0-∞)分别提高了23.60%和40.24%,作用强于维拉帕米组；④槲皮素组的Tmax与阴性组比较无显著意义,两者均大于维拉帕米组；⑤维拉帕米组和槲皮素组将Cmax分别提高了48.39%和50.26%(P<0.01)；⑥统计矩所得参数与房室模型的相应参数相近,差异均无显著性意义。上述研究结果表明,葛根素口服吸收比较快,维拉帕米和槲皮素通过抑制P-gp均可明显提高葛根素的AUC值,槲皮素还可延长MRT值。以大鼠整体动物模型进一步评价葛根素在不同剂量条件下的体内吸收药动学特征,并重点考察P-gp泵抑制剂对其吸收作用的影响,结果发现：①灌胃给药后,低剂量葛根素和高剂量葛根素的体内吸收药动学特征近似,血药浓度经时变化规律均符合二室模型,低剂量组的t1/2Ka=19.511±3.268 min,Ka=0.036±0.006min-1,高剂量组的t1／2Ka=19.956±1.737 min,Ka=0.035±0.003min-1；②维拉帕米和槲皮素组均具有明显增大吸收分数F值和显著增加AUC值的作用。维拉帕米组分别将低、高剂量葛根素的房室模型AUC(0-t)提高了11.22%和49.76%,将AUC(0-∞)提高了17.58%和41.01%；槲皮素组分别将AUC(0-t)提高了22.31%和57.46%,将AUC(0-∞)提高了9.72%和46.07%,槲皮素还可延长MRT值；维拉帕米和槲皮素组分别将小剂量葛根素(5mg／kg)的AUC(0-Tmax)提高92.09%和198.64%(P<0.01),两组还分别将大剂量葛根素(10mg／kg)的AUC(0-Tmax)提高127.61%和69.96%(P<0.05)。统计矩所得参数与房室模型的相应参数相近。③对于低剂量或高剂量葛根素,维拉帕米和槲皮素对其AUC／D比值均有提高作用,槲皮素组的比值大于维拉帕米组。维拉帕米和槲皮素对高剂量葛根素AUC／D比值的增加值(对阴性组)均分别明显高于二者对低剂量葛根素的增加值,增加2-6.5倍不等。上述研究结果表明：不同剂量葛根素在肠道吸收比较快,其吸收药动学特征相似；相同浓度的同一种P-gp泵抑制剂对高剂量葛根素吸收的提高率明显大于其对低剂量葛根素吸收的提高率。这可能与大鼠小肠黏膜上局部的P-gp泵密度有限,当被P-gp泵抑制剂显著抑制后,其对高浓度底物(葛根素)的外排相对量比低浓度底物小,故导致高浓度底物相对较多比例被吸收,提示选择P-gp泵抑制剂与底物的恰当比例对促进药物的更好吸收具有重要的意义。采用小鼠模型观察P-gp抑制剂对葛根素跨膜进入脑组织的影响,结果发现：①葛根素与维拉帕米或槲皮素配伍用药时,维拉帕米组低、高剂量组分别将脑组织的葛根素含量提高28.65%和246.56%,槲皮素组低、高剂量组则分别提高106.67%和363.82%；②维拉帕米和槲皮素促葛根素的透膜作用均具有一定的量效关系,槲皮素的作用强于维拉帕米。上述研究结果表明,维拉帕米和槲皮素通过抑制P-gp而提高葛根素在脑组织的分布量,这为葛根素与P-gp抑制剂配伍使用以提高其防治脑血管病效果提供了药动学依据。通过闭夹大脑中动脉制作局部特定区域急性脑缺血大鼠模型及缺血再灌注损伤模型,观察葛根素对大鼠急性脑缺血及缺血再灌注损伤后HSP70蛋白及Fas蛋白表达的影响,探讨葛根素保护脑缺血损伤的作用机制。结果发现：①葛根素对急性缺血性损伤的保护作用可能主要通过上调HSP70蛋白的表达而实现,与Fas蛋白的表达及细胞凋亡等相关不强；②葛根素对脑缺血再灌注损伤的保护作用除与上调HSP70表达相关外,还可能与其下调Fas蛋白的表达和减少细胞凋亡有关。结果表明,葛根素对急性脑缺血及缺血再灌注所致的脑细胞损伤均有保护作用,其保护作用机制在急性缺血性损伤期及再灌注损伤期可能因其损伤机理不同而有所不同。综上所述,槲皮素可通过抑制P-gp泵的外排作用而提高葛根素的肠道吸收速率和渗透性,促进其肠道吸收,使AUC值明显增加,从而证明槲皮素是一种高效、低毒的P-gp抑制剂,可显著提高葛根素的口服生物利用度；进一步研究发现,相同浓度的同一种P-gp泵抑制剂对高剂量葛根素吸收的提高率明显大于其对低剂量葛根素吸收的提高率,提示选择P-gp泵抑制剂与底物的恰当比例对促进药物的更好吸收具有重要的意义。此外,槲皮素还可抑制脑部的P-gp进而提高葛根素在脑组织中的分布。药效学研究表明,葛根素可通过上调HSP70蛋白的表达和下调Fas蛋白的表达而对脑缺血损伤起保护作用,但其保护作用机制在急性缺血性损伤期及再灌注损伤期可能因其损伤机理不同而有所不同。更多还原

【Abstract】 Puerarin belongs to a member of flavanoid family with polyhydroxy and can be highly effective against angiocardiopathy and cerebrovascular diseases with properties of holding pharmacokinetics of rapid absorption from the intestine and presenting in brain organ tissue across the blood brain barrier. Puerarin is a substrate of p-glycoprotein (P-gp), which is a leading cause for low permeability across intestine mucous membrane and responsible for its poor oral bioavailability due to the excretion action of p-glycoprotein from serosal to mucosal in the intestine. Intravenous injection of puerarin was widely used for clinical treatment, but it caused many severe adverse reactions and had been limited to use in clinic. To solve this problem, it is necessary to study the biological impact factors on improving oral bioavailability of puerarin by inhibition of p-glycoprotein. The traditional P-gp inhibitors may lead to various toxic effects on the patients, e.g. propafenone and verapamil damage to the heart. In order to develop an efficient but low toxic P-gp inhibitor to improve oral bioavailability of puerarin, the present paper broadly explored the effects of quercetin (another member of flavanoid family) on improving oral bioavailability of puerarin by inhibition of intestinal P-gp and on the distribution of puerarin to the brain tissue and the protecting mechanism of puerarin on the brain neurocytes of rats after ischemia brain injuries.The intestinal absorption and excretion of puerarin on the influence of the p-glycoprotein inhibitors, quercetin and verapamil were observed using rat intestinal everted gut sac in vitro. The results indicated that the three intestine segments of duodenum, jejunum and ileum had no difference in the absorption rate of puerarin without the action of any the p-glycoprotein inhibitor. Considering that the expression of p-glycoprotein in rat intestine is the highest in the ileum among the three intestine segments, we selected it as the abserved segment in our experiment of intestinal everted gut sac in vitro. Puerarin was absorbed greatly from mucosal to serosal and excreted less from serosal to mucosal in the ileum in the presence of quercetin or verapamil. The elevating percentages were 219.06%、107.29% and 60.43% in quercetin groups with high, middle and low doses, and 138.79% in verapamil group respectively. These actions were greater in the quercetin group with high dose than those in the verapamil group (P<0.01). Meanwhile these actions were observed with a dose-effect relationship in the quercetin groups.The test results of intestinal absorption of glucose showed that glucose concentration accumulated in serosal side were over 3-fold higher than that in mucosal side during the testing time course of 120min. This indicated that the testing intestinal segments were intact and active physiologically with the function of accumulation of glucose against its concentration.Absorption rate constant(Ka), effective permeability (Peff) and absorption rate(Pa%) of puerarin in each group were calculated using rat intestinal perfusion in situ model and gravimetric method. The results showed that the perfused solution dissolved with puerarin was stable with 6h, indicating that determination of puerarin was under qualified control during the testing time course, and that the values of Cout/Cin and NWF had no difference among the collecting time points in each group, which indicated that drug absorption and water absorption rates had reached equilibrium state after pre-perfusion for 1h. The parameters of Ka, Peff and Pa% were elevated significantly in the quercetin group and verapamil group respectively (P<0.01), which indicated that intestinal absorption of puerarin was obviously improved by the effects of P-gp inhibitors on intestinal absorption rate and permeability. These actions were greater in the quercetin group with high dose than those in verapamil group (P<0.01). To avoid systematic error, it is necessary to calculate these values with correct densities among groups.The results of mice model in vivo showed that plasma drug concentration of puerarin vs time conformed to two-compartment model after administration of puerarin intragastrically, with the properties of quick absorption (t1/2Ka=5.565±5.094min, Ka=0.152±0.084min-1), rapid and broad distribution to the tissues and fast elimination from the body. The absorption of puerarin in the quercetin group was greater than that in the verapamil group and this action enlarged a longer time in the former group as well. The results of statistical moment indicated that F(absorption fraction), AUC(area under the plasma concentration time curve) and MRT(mean residence time) values were increased by quercetin or verapamil respectively. The elevating percentages of AUC(0-t)s were 33.03% and 156.46% in verapamil group and quercetin group respectively, and AUC(0-∞)s were 37.04%和164.27% respectively, and AUC(0-Tmax)s were 38.57% and 110.28% in the two groups respectively. The elevating percentages of MRT(0-t) and MRT(0-∞) were 23.60% and 40.24% in quercetin group, respectively. The Cmax values were elevated by 48.39% and 50.26% in verapamil group and quercetin group respectively. The results indicated that absorption of puerarin was obviously improved by the effects of P-gp inhibitors on mice in vivo.The results of rat model in vivo indicated that the pharmacokinetics features in the puerarin group with a low dose were as similar as those in the puerarin group with a high dose, showing that plasma drug concentration of puerarin vs time conformed to two-compartment model after administration of puerarin intragastrically, with the properties of quick absorption (t1/2Ka=19.511±3.268min and Ka=0.036±0.006min-1 in the puerarin group with a low dose, t1/2Ka=19.956±1.737min and Ka=0.035±0.003min-1 in the puerarin group with a high dose), rapid and broad distribution to the tissues and fast elimination from the body. F values(absorption fraction) were greatly increased in both verapamil and quercetin groups. AUC(0-t) of verapamil group with puerarin in a low dose or in a high dose was elevated by 11.22% or 49.76%, and AUC(0-∞)s of those were elevated by 17.58% and 41.01%, respectively. AUC(0-t) of quercetin group with puerarin in a low dose or in a high dose was elevated by 22.31% or 57.46%, and AUC(0-∞)s of those were elevated by 9.72% and 46.07%, respectively. AUC(0-Tmax)s were raised by 92.09% and 198.64% respectively in verapamil group and quercetin group both with a low dose of puerarin (5mg/kg), and were raised by 127.61% and 69.96% respectively in the two groups with a high dose of puerarin (10mg/kg). The AUC values were significantly increased in both quercetin and verapamil groups and MRT value was increased by the quercetin group as well.To compare the difference between the two negative groups with puerarin in low or high dose respectively and compare the difference between the two groups exerted by the same P-gp inhibitor in the same dose, we observed the effects of inhibitors on AUC/D(dose) values of puerarin with different doses. The results showed that quercetin and verapamil could both increase the AUC/D value either in the group with puerarin in low or high dose, but the increasing rate in the the group with puerarin in high dose was greater than that in low dose, from 2-6.5 fold. These indicate that the absorption rate of puerarin with high dose were greater than that of puerarin with low dose under the same influence of P-gp inhibitor. This fact can be explained by the reason that the excretion rate of puerarin in high dose is relatively less than that of puerarin in low dose because of full occupation of P-gp receptor in local intestinal mucosa, indicating the significance to chose a suitable rate of P-gp receptor to the substrate puerarin.P-glycoprotein expresses highly on the contacting interface between endothelial cell of cerebullar blood circulation and periphery blood circulation, which plays an important role in inhibiting some P-gp sunstrates transportation to the brain tissue. Thus inhibition of P-gp could have beem considered to be an effective route to improve the P-gp sunstrates across cerebullar membrane and treat cerebrovascular diseases efficiently. The presencent study observed the distribution of puerarin to brain tissues of mice under the influence of the two P-gp inhibitors and the results showed that either quercetin or verapamil could increase the quantity of puerarin transported to the brain tissues. The elevating percentages were 28.65% and 246.56% in verapamil groups with a low dose and a high dose and the elevating percentages were 106.67% and 363.82% in quercetin groups with a low dose and a high dose respectively. The two P-gp inhibitors had dose-effect relationship respectively and the action of quercetin was stronger than that of verapamil. The results provide the experimental pharmacokinetics data for the potential treatment of cerebrovascular diseases with the comitant use of puerarin and its P-gp sunstrate.The present study was designed to investigate the possible properties of the injured brain neurocytes, the expression of HSP70 (heat shock protein70) and Fas protein after acute local ischemia brain injury and local cerebral ischemia-reperfusion injury in rats and to investigate the protecting mechanism of puerarin on the brain neurocytes of rats in acute local ischemia brain injury and local cerebral ischemia-reperfusion injury using rat models of acute local cerebral ischemia and ischemia-reperfusion made by ligatting the middle cerebral artery.The results showed that puerarin had the protective actions in both processes of acute cerebral ischemia and cerebral ischemia-reperfusion. The protecting mechanism varied with the difference of neurocyte injuries:1. In the process of acute cerebral ischemia, the protecting effect of puerarin mainly related to up-regulation of HSP70 expression, while not correlated to the expression of Fas protein.2. In the period of cerebral ischemia-reperfusion, the protecting effect of puerarin related to both processes of up-regulation of HSP70 expression and down regulation of Fas expression.3. The neuroprotective effects of puerarin in the duration of acute cerebral ischemia exerted the actions of improving the level of stress and promoting the recovery of degenerated protein. During cerebral ischemia-reperfusion, puerarin acted on not only these effects, but reducing neurocyte apoptosis.Summarily, the effects of quercetin on improving oral bioavailability of puerarin are associated with its inhibition of intestinal P-gp, which elevates absorption rate and permeability of puerarin across the intestine and enhances its AUC values in rats’ plasma. These indicate that quercetin is an efficient and low toxic P-gp inhibitor to improve oral bioavailability of puerarin. Furthermore, the identical P-gp inhibitor with the same dose could both increase the AUC/D value either in the group with puerarin in a low or high dose, but the increasing rate in the the group with puerarin in a high dose was greater than that in a low dose, indicating the importance of selecting a suitable ratio between P-gp inhibitor and its substrate in improving oral bioavailability of puerarin. In addition to these action, quercetin could increase the quantity of puerarin transported to the brain tissues, providing the experimental pharmacokinetics data for the potential treatment of cerebrovascular diseases with the comitant use of puerarin and its P-gp sunstrate. The results of pharmacodynamics study showed that puerarin had the protective actions in both processes of acute cerebral ischemia and cerebral ischemia-reperfusion. The protecting mechanism related to up-regulation of HSP70 expression and/or down regulation of Fas expression, which varied with the periods of cerebral ischemia and ischemia-reperfusion injuries and the difference of neurocyte injuries.更多还原