Studies On The Aborptive Kinetics Of Puerarin And Its Protecting Mechanism On Cerebral Ischemia Injuries

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.